Thermal cycler for automatic performance of the polymerase chain reaction with close temperature control

ABSTRACT

An instrument for performing highly accurate PCR employing a sample block in microtiter tray format. The sample block has local balance and local symmetry. A three zone film heater controlled by a computer and ramp cooling solenoid valves also controlled by the computer for gating coolant flow through the block controls the block temperature. Constant bias cooling is used for small changes. Sample temperature is calculated instead of measured. A platen deforms plastic caps to apply a minimum acceptable threshold force for seating the tubes and thermally isolates them. A cover isolates the block. The control software includes diagnostics. An install program tests and characterizes the instrument. A new user interface is used. Disposable, multipiece plastic microtiter trays to give individual freedom to sample tubes are taught.

This is a continuation-in-part application of U.S. patent applicationSer. No. 07/620,606, filed Nov. 29, 1990 by Mossa et al. and U.S. patentapplication Ser. No. 07/670,545, filed Mar. 14, 1991, both of which arehereby incorporated herein by reference both abandoned. MicroficheAppendicies C-F are attached, including 10 sheets of microfichecomprising 890 frames.

The invention pertains to the field of computer directed instruments forperforming the polymerase chain reaction (hereafter PCR). Moreparticularly, the invention pertains to automated instruments that canperform the polymerase chain reaction simultaneously on many sampleswith a very high degree of precision as to results obtained for eachsample. This high precision provides the capability, among other things,of performing so-called "quantitative PCR".

To amplify DNA (Deoxyribose Nucleic Acid) using the PCR process, it isnecessary to cycle a specially constituted liquid reaction mixturethrough a PCR protocol including several different temperatureincubation periods. The reaction mixture is comprised of variouscomponents such as the DNA to be amplified and at least two primersselected in a predetermined way so as to be sufficiently complementaryto the sample DNA as to be able to create extension products of the DNAto be amplified. The reaction mixture includes various enzymes and/orother reagents, as well as several deoxyribonucleoside triphosphatessuch as dATP, dCTP, dGTP and dTTP. Generally, the primers areoligonucleotides which are capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complimentary to a nucleic acid strand isinduced, i.e., in the presence of nucleotides and inducing agents suchas thermostable DNA polymerase at a suitable temperature and pH.

The Polymerase Chain Reaction (PCR) has proven a phenomenally successfultechnology for genetic analysis, largely because it is so simple andrequires relatively low cost instrumentation. A key to PCR is theconcept of thermocycling: alternating steps of melting DNA, annealingshort primers to the resulting single strands, and extending thoseprimers to make new copies of double stranded DNA. In thermocycling, thePCR reaction mixture is repeatedly cycled from high temperatures (>90°C.) for melting the DNA, to lower temperatures (40° C. to 70° C.) forprimer annealing and extension. The first commercial system forperforming the thermal cycling required in the polymerase chainreaction, the Perkin-Elmer Cetus DNA Thermal Cycler, was introduced in1987.

Applications of PCR technology are now moving from basic research toapplications in which large numbers of similar amplifications areroutinely run. These areas include diagnostic research,biopharmaceutical development, genetic analysis, and environmentaltesting. Users in these areas would benefit from a high performance PCRsystem that would provide the user with high throughput, rapidturn-around time, and reproducible results. Users in these areas must beassured of reproducibility from sample-to-sample, run-to-run,lab-to-lab, and instrument-to-instrument.

For example, the physical mapping process in the Human Genome Projectmay become greatly simplified by utilizing sequence tagged sites. An STSis a short, unique sequence easily amplified by PCR and which identifiesa location on the chromosome. Checking for such sites to make genomemaps requires amplifying large numbers of samples in a short time withprotocols which can be reproducibly run throughout the world.

As the number of PCR samples increases, it becomes more important tointegrate amplification with sample preparation and post-amplificationanalysis. The sample vessels must not only allow rapid thermal cyclingbut also permit more automated handling for operations such as solventextractions and centrifugation. The vessels should work consistently atlow volumes, to reduce reagent costs.

Generally PCR temperature cycling involves at least two incubations atdifferent temperatures. One of these incubations is for primerhybridization and a catalyzed primer extension reaction. The otherincubation is for denaturation, i.e., separation of the double strandedextension products into single strand templates for use in the nexthybridization and extension incubation interval. The details of thepolymerase chain reaction, the temperature cycling and reactionconditions necessary for PCR as well as the various reagents and enzymesnecessary to perform the reaction are described in U.S. Pat. Nos.4,683,202, 4,683,195, EPO Publication 258,017 and U.S. Pat. No.4,889,818 (Taq polymerase enzyme patent), which are hereby incorporatedby reference.

The purpose of a polymerase chain reaction is to manufacture a largevolume of DNA which is identical to an initially supplied small volumeof "seed" DNA. The reaction involves copying the strands of the DNA andthen using the copies to generate other copies in subsequent cycles.Under ideal conditions, each cycle will double the amount of DNA presentthereby resulting in a geometric progression in the volume of copies ofthe "target" or "seed" DNA strands present in the reaction mixture.

A typical PCR temperature cycle requires that the reaction mixture beheld accurately at each incubation temperature for a prescribed time andthat the identical cycle or a similar cycle be repeated many times. Atypical PCR program starts at a sample temperature of 94° C. held for 30seconds to denature the reaction mixture. Then, the temperature of thereaction mixture is lowered to 37° C. and held for one minute to permitprimer hybridization. Next, the temperature of the reaction mixture israised to a temperature in the range from 50° C. to 72° C. where it isheld for two minutes to promote the synthesis of extension products.This completes one cycle. The next PCR cycle then starts by raising thetemperature of the reaction mixture to 94° C. again for strandseparation of the extension products formed in the previous cycle(denaturation). Typically, the cycle is repeated 25 to 30 times.

Generally, it is desirable to change the sample temperature to the nexttemperature in the cycle as rapidly as possible for several reasons.First, the chemical reaction has an optimum temperature for each of itsstages. Thus, less time spent at nonoptimum temperatures means a betterchemical result is achieved. Another reason is that a minimum time forholding the reaction mixture at each incubation temperature is requiredafter each said incubation temperature is reached. These minimumincubation times establish the "floor" or minimum time it takes tocomplete a cycle. Any time transitioning between sample incubationtemperatures is time which is added to this minimum cycle time. Sincethe number of cycles is fairly large, this additional time unnecessarilylengthens the total time needed to complete the amplification.

In some prior automated PCR instruments, the reaction mixture was storedin a disposable plastic tube which is closed with a cap. A typicalsample volume for such tubes was approximately 100 microliters.Typically, such instruments used many such tubes filled with sample DNAand reaction mixture inserted into holes called sample wells in a metalblock. To perform the PCR process, the temperature of the metal blockwas controlled according to prescribed temperatures and times specifiedby the user in a PCR protocol file. A computer and associatedelectronics then controlled the temperature of the metal block inaccordance with the user supplied data in the PCR protocol file definingthe times, temperatures and number of cycles, etc. As the metal blockchanged temperature, the samples in the various tubes followed withsimilar changes in temperature. However, in these prior art instrumentsnot all samples experienced exactly the same temperature cycle. In theseprior art PCR instruments, errors in sample temperature were generatedby nonuniformity of temperature from place to place within the metalsample block, i.e., temperature gradients existed within the metal ofthe block thereby causing some samples to have different temperaturesthan other samples at particular times in the cycle. Further, there weredelays in transferring heat from the sample block to the sample, but thedelays were not the same for all samples. To perform the PCR processsuccessfully and efficiently, and to enable so called "quantitative"PCR, these time delays and temperature errors must be minimized to agreat extent.

The problems of minimizing time delays for heat transfer to and from thesample liquid and minimizing temperature errors due to temperaturegradients or nonuniformity in temperature at various points on the metalblock become particularly acute when the size of the region containingsamples becomes large. It is a highly desirable attribute for a PCRinstrument to have a metal block which is large enough to accommodate 96sample tubes arranged in the format of an industry standard microtiterplate.

The microtiter plate is a widely used means for handling, processing andanalyzing large numbers of small samples in the biochemistry andbiotechnology fields. Typically, a microtiter plate is a tray which is35/8 inches wide and 5 inches long and contains 96 identical samplewells in an 8 well by 12 well rectangular array on 9 millimeter centers.Although microtiter plates are available in a wide variety of materials,shapes and volumes of the sample wells, which are optimized for manydifferent uses, all microtiter plates have the same overall outsidedimensions and the same 8×12 array of wells on 9 millimeter centers. Awide variety of equipment is available for automating the handling,processing and analyzing of samples in this standard microtiter plateformat.

Generally microtiter plates are made of injection molded or vacuumformed plastic and are inexpensive and considered disposable.Disposability is a highly desirable characteristic because of the legalliability arising out of cross contamination and the difficulty ofwashing and drying microtiter plates after use.

It is therefore a highly desirable characteristic for a PCR instrumentto be able to perform the PCR reaction on up to 96 samplessimultaneously said samples being arranged in a microtiter plate format.

Of course, the size of the metal block which is necessary to heat andcool 96 samples in an 8×12 well array on 9 millimeter centers is fairlylarge. This large area block creates multiple challenging engineeringproblems for the design of a PCR instrument which is capable of heatingand cooling such a block very rapidly in a temperature range generallyfrom 0° to 100° C. with very little tolerance for temperature variationsbetween samples. These problems arise from several sources. First, thelarge thermal mass of the block makes it difficult to move the blocktemperature up and down in the operating range with great rapidity.Second, the need to attach the block to various external devices such asmanifolds for supply and withdrawal of cooling liquid, block supportattachment points, and associated other peripheral equipment creates thepotential for temperature gradients to exist across the block whichexceed tolerable limits.

There are also numerous other conflicts between the requirements in thedesign of a thermal cycling system for automated performance of the PCRreaction or other reactions requiring rapid, accurate temperaturecycling of a large number of samples. For example, to change thetemperature of a metal block rapidly, a large amount of heat must beadded to, or removed from the sample block in a short period of time.Heat can be added from electrical resistance heaters or by flowing aheated fluid in contact with the block. Heat can be removed rapidly byflowing a chilled fluid in contact with the block. However, it isseemingly impossible to add or remove large amounts of heat rapidly in ametal block by these means without causing large differences intemperature from place to place in the block thereby forming temperaturegradients which can result in nonuniformity of temperature among thesamples.

Even after the process of addition or removal of heat is terminated,temperature gradients can persist for a time roughly proportional to thesquare of the distance that the heat stored in various points in theblock must travel to cooler regions to eliminate the temperaturegradient. Thus, as a metal block is made larger to accommodate moresamples, the time it takes for temperature gradients existing in theblock to decay after a temperature change causes temperature gradientswhich extend across the largest dimensions of the block can becomemarkedly longer. This makes it increasingly difficult to cycle thetemperature of the sample block rapidly while maintaining accuratetemperature uniformity among all the samples.

Because of the time required for temperature gradients to dissipate, animportant need has arisen in the design of a high performance PCRinstrument to prevent the creation of temperature gradients that extendover large distances in the block. Another need is to avoid, as much aspossible, the requirement for heat to travel across mechanicalboundaries between metal parts or other peripheral equipment attached tothe block. It is difficult to join metal parts in a way that insuresuniformly high thermal conductance everywhere across the joint.Nonuniformities of thermal conductance will generate unwantedtemperature gradients.

SUMMARY OF THE INVENTION

According to the teachings of the invention, there is disclosed herein athin walled sample tube for decreasing the delay between changes insample temperature of the sample block and corresponding changes intemperature of the reaction mixture. Two different sample tube sizes aredisclosed, but each has a thin walled conical section that fits into amatching conical recess in the sample block. Typically, cones with 17°angles relative to the longitudinal axis are used to prevent jamming ofthe tubes into the sample block but to allow snug fit. Other shapes andangles would also suffice for purposes of practicing the invention.

Also, other types of heat exchangers can also be used other than sampleblocks such as liquid baths, ovens, etc. However, the wall thickness ofthe section of the sample tube which is in contact with whatever heatexchange is being used should be as thin as possible so long as it issufficiently strong to withstand the thermal stresses of PCR cycling andthe stresses of normal use. Typically, the sample tubes are made ofautoclavable polypropylene such as Himont PD701 with a wall thickness ofthe conical section in the range from 0.009 to 0.012 inches plus orminus 0.001 inches. Most preferably, the wall thickness is 0.012 inchesfor larger tubes as shown in FIG. 50.

In the preferred embodiment, the sample tube also has a thicker walledcylindrical section which joins with the conical section. Thiscylindrical section provides containment for the original reactionmixture or reagents which may be added after PCR processing.

The sample tube shown in FIG. 50 has industry standard configurationexcept for the thin walls for compatibility in other PCR systems. Thesample tube of FIG. 15 is a shorter tube which can be used with thesystem disclosed herein. The other subject matter of the systemenvironment in which use of the thin walled sample tubes is preferredare summarized below.

There is also described herein a novel method and apparatus forachieving very accurate temperature control for a very large number ofsamples arranged in the microtiter plate format during the performanceof very rapid temperature cycling PCR protocols. The teachings of theinvention contemplate a novel structure for a sample block, sample tubesand supporting mounting, heating and cooling apparatus, controlelectronics and software, a novel user interface and a novel method ofusing said apparatus to perform the PCR protocol.

The instrument described herein is designed to do PCR gene amplificationon up to 96 samples with very tight tolerances of temperature controlacross the universe of samples. This means that all samples go up anddown in temperature simultaneously with very little difference intemperature between different wells containing different samples, thisbeing true throughout the polymerase chain reaction cycle. Theinstrument described herein is also capable of very tight control of thereaction mixture concentration through control of the evaporation andcondensation processes in each sample well. Further, the instrumentdescribed herein is capable of processing up to 96 samples of 100microliters each from different donor sources with substantially nocross-contamination between sample wells.

The teachings of the invention herein include a novel method of heatingand cooling an aluminum sample block to thermally cycle samples in thestandard 96-well microtiter plate format with the result that excellentsample-to-sample uniformity exists despite rapid thermal cycling rates,noncontrolled varying ambient temperatures and variations in otheroperating conditions such as power line voltage and coolanttemperatures.

The teachings of the invention also contemplate a novel design for adisposable plastic 96-well microtiter plate for accommodation of up to96 individual sample tubes containing DNA for thermal cycling eachsample tube having individual freedom of movement sufficient to find thebest fit with the sample block under downward pressure from a heatedcover. The microtiter plate design, by allowing each tube to find thebest fit, provides high and uniform thermal conductance from the sampleblock to each sample tube even if differing rates of thermal expansionand contraction between the metal of the block and the plastic of thesample tube and microtiter plate structure cause the relativecenter-to-center dimensions of the wells in the sample block to changerelative to the center-to-center distance of the sample tubes in thedisposable microtiter plate structure.

The teachings of the invention also contemplate a novel method andapparatus for controlling the PCR instrument which includes the abilityto continuously calculate and display the temperature of the samplesbeing processed without directly measuring these temperatures. Thesecalculated temperatures are used to control the time that the samplesare held within the given temperature tolerance band for each targettemperature of incubation. The control system also controls a three-zoneheater thermally coupled to the sample block and gates fluid flowthrough directionally interlaced ramp cooling channels in the sampleblock which, when combined with a constant bias cooling flow of coolantthrough the sample block provides a facility to achieve rapidtemperature changes to and precise temperature control at targettemperatures specified by the user. The method and apparatus forcontrolling the three-zone heater includes an apparatus for taking intoaccount, among other things, the line voltage, block temperature,coolant temperature and ambient temperature in calculating the amount ofelectrical energy to be supplied to the various zones of the three-zoneheater. This heater has zones which are separately controllable underthe edges or "guard bands" of the sample block so that excess heatlosses to the ambient through peripheral equipment attached to the edgesof the sample block can be compensated. This helps prevent thermalgradients from forming.

The teachings of the invention also contemplate a novel method andapparatus for preventing loss of solvent from the reaction mixtures whenthe samples are being incubated at temperatures near their boilingpoint. A heated platen covers the tops of the sample tubes and is incontact with an individual cap which provides a gas-tight seal for eachsample tube. The heat from the platen heats the upper parts of eachsample tube and the cap to a temperature above the condensation pointsuch that no condensation and refluxing occurs within any sample tube.Condensation represents a relatively large heat transfer since an amountof heat equal to the heat of vaporization is given up when water vaporcondenses. This could cause large temperature variations from sample tosample if the condensation does not occur uniformly. The heated platenprevents any condensation from occurring in any sample tube therebyminimizing this source of potential temperature errors. The use of theheated platen also reduces reagent consumption.

Furthermore, the heated platen provides a downward force for each sampletube which exceeds an experimentally determined minimum downward forcenecessary to keep all sample tubes pressed firmly into the temperaturecontrolled sample block so as to establish and maintain uniformblock-to-tube thermal conductance for each tube. This uniformity ofthermal conductance is established regardless of variations from tube totube in length, diameter, angle or other dimensional errors whichotherwise could cause some sample tubes to fit more snugly in theircorresponding sample wells than other sample tubes.

The heated platen softens the plastic of each cap but does not totallydestroy the cap's elasticity. Thus, a minimum threshold downward forcedis successfully applied to each tube despite differences in tube heightfrom tube to tube.

The PCR instrument described herein reduces cycle times by a factor of 2or more and lowers reagent cost by accommodating PCR volumes down to 20ul but remains compatible with the industry standard 0.5 mlmicrocentrifuge tube.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of the thermal cycler according to theteachings of the invention.

FIG. 2 is a plan view of a sample block according to the teachings ofthe invention.

FIG. 3 is a side, elevation view of the sample block showing the biasand ramp cooling channels.

FIGS. 4 and 5 are end, elevation views of the sample block.

FIG. 4.1 is an elevation view of the edge of an alternate sample block.

FIG. 6 is a sectional view of the sample block taken along section line6--6' in FIG. 2.

FIG. 7 is a sectional view of the sample block taken along section line7--7' in FIG. 2.

FIG. 8 is a sectional view of the sample block taken along section line8--8' in FIG. 2

FIG. 9 is a cross-sectional, elevation view of the sample blockstructure after assembly with the three-zone film heater and blocksupport.

FIG. 10 is a graph of power line voltage illustrating the form of powercontrol to the three-zone film heater.

FIG. 11 is a temperature graph showing a typical three incubationtemperature PCR protocol.

FIG. 12 is a cross-sectional view of the sample block illustrating thelocal zone concept.

FIG. 13 is a plan view of the three-zone heater.

FIG. 14 is a graph of sample temperature versus time illustrating theeffect of an τ of a sample tube seating force F which is too low.

FIG. 14.1 is a graph of sample temperature versus time illustrating boththe effect of an τ of a sample tube seating force F which is too low,and the block temperature overshoot effectuated by version 2 of thecontrol software.

FIG. 15 is a cross-sectional view of a sample tube and cap seated in thesample block.

FIG. 16A is a graph of the impulse response of an RC circuit.

FIG. 16B is a graph of an impulse excitation pulse.

FIG. 16C is a graph illustrating how the convolution of the thermalimpulse response and the temperature history of the block give thecalculated sample temperature.

FIG. 16D illustrates the electrical analog of the thermal response ofthe sample block/sample tube system.

FIG. 17 illustrates how the calculated temperatures of six differentsamples all converge on a target temperature to within about 0.5° C. ofeach other when the constants of proportionality for the equations usedto control the three zone heater are properly set.

FIG. 18 is a graph illustrating how the denaturation target temperatureaffects the amount of DNA generated.

FIG. 19 is a cross-sectional view of the sliding cover and heatedplaten.

FIG. 20 is a perspective view of the sliding cover, sample block and theknob used to lower the heated platen.

FIG. 21A is a cross-sectional view of the assembly of one embodiment ofthe frame, retainer, sample tube and cap when seated on a sample block.

FIG. 21B is a cross-sectional view of the assembly of the preferredembodiment of the frame, retainer, sample tube and cap when seated onthe sample block.

FIG. 22 is a top, plan view of the plastic, disposable frame for themicrotiter plate.

FIG. 23 is a bottom, plan view of the frame.

FIG. 24 is an end, elevation view of the frame.

FIG. 25 is another end, elevation view of the frame.

FIG. 26 is a cross-sectional view of the frame taken along section line26--26' in FIG. 22.

FIG. 27 is a cross-sectional view of the frame taken along section line27--27' in FIG. 22.

FIG. 28 is an edge elevation view and partial section of the frame.

FIG. 29 is a sectional view of the preferred sample tube.

FIG. 30 is a sectional view of the upper part of the sample tube.

FIG. 31 is an elevation view of a portion of the cap strip.

FIG. 32 is a top view of a portion of the cap strip.

FIG. 33 is a top, plan view of the plastic, disposable retainer portionof the 96 well microtiter tray.

FIG. 34 is a side, elevation view with a partial section of theretainer.

FIG. 35 is an end, elevation view of the retainer.

FIG. 36 is a sectional view of the retainer taken along section line36--36' in FIG. 33.

FIG. 37 is a sectional view of the retainer taken along section line37-37' in FIG. 33.

FIG. 38 is a plan view of the plastic disposable support base of the 96well microtiter tray.

FIG. 39 is a bottom plan view of the base.

FIG. 40 is a side elevation view of the base.

FIG. 41 is an end elevation view of the base.

FIG. 42 is a sectional view of the support base taken along section line42-42' in FIG. 38.

FIG. 43 is a sectional view of the support base taken along section line43-43' in FIG. 38.

FIG. 44 is a section view of the base taken along section line 44-44' inFIG. 38.

FIG. 45 is a perspective exploded view of the plastic disposable itemsthat comprise the microtiter tray with some sample tubes and caps inplace.

FIG. 46 is a diagram of the coolant control system 24 in FIG. 1.

FIGS. 47A and 47B are a block diagram of the control electronicsaccording to the teachings of the invention.

FIG. 48 is a schematic of a typical zener temperature sensor.

FIG. 49 is a time line diagram of a typical sample period.

FIG. 50 is elevation sectional view of a tall thin walled sample tubemarketed under the trademark MAXIAMP.

FIG. 51 is a graph showing the difference in response time between thethin walled sample tubes and the thick walled prior art tubes.

FIG. 52 is a plan view of a sample tube and cap.

FIGS. 53 and 54 are flow charts of the power up test sequence.

FIG. 55 is a flow diagram representing the Power-Up sequence as it isimplemented in Version 2 of the Electronics and Software.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIG. 1 there is shown a block diagram of the major systemcomponents of one embodiment of a computer directed instrument forperforming PCR according to the teachings of the invention. Samplemixtures including the DNA or RNA to be amplified are placed in thetemperature-programmed sample block 12 and are covered by heated cover14.

A user supplies data defining time and temperature parameters of thedesired PCR protocol via a terminal 16 including a keyboard and display.The keyboard and display are coupled via bus 18 to a control computer 20(hereafter sometimes referred to as a central processing unit or CPU).This central processing unit 20 includes memory which stores the controlprogram described below, the data defining the desired PCR protocol andcertain calibration constants described below. The control programcauses the CPU 20 to control temperature cycling of the sample block 12and implements a user interface which provides certain displays to theuser and which receives data entered by the user via the keyboard of theterminal 16.

In the preferred embodiment, the central processing unit 20 is customdesigned. The wiring diagrams for the CPU and support electronics isgiven in Microfiche Appendix E. The actual control program is givenbelow in Microfiche Appendix C (Version 2 of the control program isincluded as Microfiche Appendix F). A block diagram of the electronicswill be discussed in more detail below. In alternative embodiments, thecentral processing unit 20 and associated peripheral electronics tocontrol the various heaters and other electro-mechanical systems of theinstrument and read various sensors could be any general purposecomputer such as a suitably programmed personal computer ormicrocomputer.

The samples 10 are stored in capped disposable tubes which are seated inthe sample block 12 and are thermally isolated from the ambient air by aheated cover 14 which contacts a plastic disposable tray to be describedbelow to form a heated, enclosed box in which the sample tubes reside.The heated cover serves, among other things, to reduce undesired heattransfers to and from the sample mixture by evaporation, condensationand refluxing inside the sample tubes. It also reduces the chance ofcross contamination by keeping the insides of the caps dry therebypreventing aerosol formation when the tubes are uncapped. The heatedcover is in contact with the sample tube caps and keeps them heated to atemperature of approximately 104° C. or above the condensation points ofthe various components of the reaction mixture.

The central processing unit 20 includes appropriate electronics to sensethe temperature of the heated cover 14 and control electric resistanceheaters therein to maintain the cover 14 at a predetermined temperature.Sensing of the temperature of the heated cover 14 and control of theresistance heaters therein is accomplished via a temperature sensor (notshown) and bus 22.

A coolant control system 24 continuously circulates a chilled liquidcoolant such as a mixture of automobile antifreeze and water throughbias cooling channels (not shown) in the sample block 12 via input tubes26 and output tube 28. The coolant control system 24 also controls fluidflow through higher volume ramp cooling fluid flow paths (not shown) inthe sample block 12. The ramp cooling channels are used to rapidlychange the temperature of the sample block 12 by pumping large volumesof chilled liquid coolant through the block at a relatively high flowrate. Ramp cooling liquid coolant enters the sample block 12 throughtube 30 and exits the sample block through tube 32. The details of thecoolant control system are shown in FIG. 46. The coolant control systemwill be discussed more fully below in the description of the electronicsand software of the control system.

Typically, the liquid coolant used to chill the sample block 12 consistsmainly of a mixture of water and ethylene glycol. The liquid coolant ischilled by a heat exchanger 34 which receives liquid coolant which hasextracted heat from the sample block 12 via input tube 36. The heatexchanger 34 receives compressed liquid freon refrigerant via input tube38 from a refrigeration unit 40. This refrigeration unit 40 includes acompressor (not shown), a fan 42 and a fin tube heat radiator 44. Therefrigeration unit 40 compresses freon gas received from the heatexchanger 34 via tube 46. The gaseous freon is cooled and condensed to aliquid in the fin tube condenser 44. The pressure of the liquid freon ismaintained above its vapor pressure in the fin tube condenser by a flowrestrictor capillary tube 47. The output of this capillary tube iscoupled to the input of the heat exchanger 34 via tube 38. In the heatexchanger, the pressure of the freon is allowed to drop below the freonvapor pressure, and the freon expands. In this process of expansion,heat is absorbed from the warmed liquid coolant circulating in the heatexchanger and this heat is transferred to the freon thereby causing thefreon to boil. The warmed freon is then extracted from the heatexchanger via tube 46 and is compressed and again circulated through thefin tube condenser 44. The fan 42 blows air through the fin tubecondenser 44 to cause heat in the freon from tube 46 to be exchangedwith the ambient air. As symbolized by arrows 48. The refrigeration unit40 should be capable of extracting 400 watts of heat at 30° C. and 100watts of heat at 10° C. from the liquid coolant to support the rapidtemperature cycling according to the teachings of the invention.

In the preferred embodiment, the apparatus of FIG. 1 is enclosed withina housing (not shown). The heat 48 expelled to the ambient air is keptwithin the housing to aid in evaporation of any condensation whichoccurs on the various tubes carrying chilled liquid coolant or freonfrom one place to another. This condensation can cause corrosion ofmetals used in the construction of the unit or the electronic circuitryand should be removed. Expelling the heat 48 inside the enclosure helpsevaporate any condensation to prevent corrosion.

After exchanging its heat with the freon, the liquid coolant exits theheat exchanger 34 via tube 50 and reenters the coolant control systemwhere it is gated as needed to the sample block during rapid coolingportions of the PCR cycle defined by data entered by the user viaterminal 16.

As noted above, the PCR protocol involves incubations at at least twodifferent temperatures and often three different temperatures. A typicalPCR cycle is shown in FIG. 11 with a denaturation incubation 170 done ata temperature near 94° C., a hybridization incubation 122 done at atemperature near room temperature (25° C.-37° C.) and an extensionincubation 174 done at a temperature near 50° C. These temperatures aresubstantially different, and, therefore means must be provided to movethe temperature of the reaction mixture of all the samples rapidly fromone temperature to another. The ramp cooling system is the means bywhich the temperature of the sample block 12 is brought down rapidlyfrom the high temperature denaturation incubation to the lowertemperature hybridization and extension incubation temperatures.Typically the coolant temperature is in the range from 10°-20° C. Whenthe coolant is at 20° C. it can pump out about 400 watts of heat fromthe sample block. Typically the ramp cooling channel dimensions, coolanttemperature and coolant flow rate are set such that peak cooling of5°-6° C. per second can be achieved near the high end of the operatingrange (100° C.) and an average cooling rate of 2.5° C. per second isachieved in bringing the sample block temperature down from 94° C. to37° C.

The ramp cooling system, in some embodiments, may also be used tomaintain the sample block temperature at or near the target incubationtemperature also. However, in the preferred embodiment, smalltemperature changes of the sample block 12 in the downward direction tomaintain target incubation temperature are implemented by the biascooling system.

As seen in FIG. 46, a pump 41 constantly pumps coolant from afilter/reservoir 39 (130 milliliter capacity) via 1/2" pipe and pumps itvia a 1/2" pipe to a branching intersection 47. The pump 41 suppliescoolant to pipe 45 at a constant flow rate of 1-1.3 gallons per minute.At the intersection 47, a portion of the flow in tube 45 is diverted asthe constant flow through the bias cooling channels 49. Another portionof the flow in tube 45 is diverted through a flow restrictor 51 tooutput tube 38. Flow restrictor 51 maintains sufficient pressure in thesystem such that a positive pressure exists at the input 53 of a twostate solenoid operated valve 55 under the control of the CPU 20 via bus54. When ramp cooling is desired to implement a rapid downwardtemperature change, the CPU 20 causes the solenoid operated valve 55 toopen to allow flow of coolant through the ramp cooling channels 57.There are 8 ramp cooling channels so the flow rate through each rampcooling channel is about 1/8 gallon per minute. The flow rate throughthe bias cooling channels is much less because of the greatly restrictedcross-sectional area thereof.

The bias cooling system provides a small constant flow of chilledcoolant through bias cooling channels 49 in the sample block 12. Thiscauses a constant, small heat loss from the sample block 12 which iscompensated by a multi-zone heater 156 which is thermally coupled to thesample block 12 for incubation segments where the temperature of thesample block is to maintained at a steady value. The constant small heatloss caused by the bias cooling flow allows the control system toimplement proportional control both upward and downward in temperaturefor small temperatures. This means both heating and cooling atcontrolled, predictable, small rates is available to the temperatureservo system to correct for block temperature errors to cause the blocktemperature to faithfully track a PCR temperature profile entered by theuser. The alternative would be to cut off power to the film heater andallow the sample block to cool by giving up heat to the ambient byradiation and convection when the block temperature got too high. Thiswould be too slow and too unpredictable to meet tight temperaturecontrol specifications for quantitative PCR cycling.

This multi-zone heater 156 is controlled by the CPU 20 via bus 52 inFIG. 1 and is the means by which the temperature of the sample block 12is raised rapidly to higher incubation temperatures from lowerincubation temperatures and is the means by which bias cooling iscompensated and temperature errors are corrected in the upward directionduring temperature tracking and control during incubations.

In alternative embodiments, bias cooling may be eliminated or may besupplied by other means such as by the use of a cooling fan and coolingfins formed in the metal of the sample block, peltier junctions orconstantly circulating tap water. Care must be taken however in thesealternative embodiments to insure that temperature gradients are notcreated in the sample block which would cause the temperature of somesamples to diverge from the temperature of other samples therebypossibly causing different PCR amplification results in some sampletubes than in others. In the preferred embodiment, the bias cooling isproportional to the difference between the block temperature and thecoolant temperature.

The CPU 20 controls the temperature of the sample block 12 by sensingthe temperature of the metal of the sample block via temperature sensor21 and bus 52 in FIG. 1 and by sensing the temperature of thecirculating coolant liquid via bus 54 and a temperature sensor in thecoolant control system. The temperature sensor for the coolant is shownat 61 in FIG. 46. The CPU also senses the internal ambient airtemperature within the housing of the system via an ambient airtemperature sensor 56 in FIG. 1. Further, the CPU 20 senses the linevoltage for the input power on line 58 via a sensor symbolized at 63.All these items of data together with items of data entered by the userto define the desired PCR protocol such as target temperatures and timesfor incubations are used by a control program to be described in moredetail below. This control program calculates the amount of power toapply to the various zones of the multi-zone sample block film heater156 via the bus 52 and generates a coolant control signal to open orclose the solenoid operated valve 55 in the coolant control system 24via bus 54 so as to cause the temperature of the sample block to followthe PCR protocol defined by data entered by the user.

Referring to FIG. 2, there is shown a top view of the sample block 12.The purpose of the sample block 12 is to provide a mechanical supportand heat exchange element for an array of thin walled sample tubes whereheat may be exchanged between the sample liquid in each sample tube andliquid coolant flowing in the bias cooling and ramp cooling channelsformed in the sample block 12. Further, it is the function of the sampleblock 12 to provide this heat exchange function without creating largetemperature gradients between various ones of the sample wells such thatall sample mixtures in the array experience the same PCR cycle eventhough they are spatially separated. It is an overall objective of thePCR instrument described herein to provide very tight temperaturecontrol over the temperature of the sample liquid for a plurality ofsamples such that the temperature of any sample liquid does not varyappreciably (approximately plus or minus 0.5° C.) from the temperatureof any other sample liquid in another well at any point in the PCRcycle.

There is an emerging branch of PCR technology called "quantitative" PCR.In this technology, the objective is to perform PCR amplification asprecisely as possible by causing the amount of target DNA to exactlydouble on every cycle. Exact doubling on every cycle is difficult orimpossible to achieve but tight temperature control helps.

There are many sources of errors which can cause a failure of a PCRcycle to exactly double the amount of target DNA (hereafter DNA shouldbe understood as also referring to RNA) during a cycle. For example, insome PCR amplifications, the process starts with a single cell of targetDNA. An error that can easily occur results when this single cell sticksto the wall of the sample tube and does not amplify in the first severalcycles.

Another type of error is the entry of a foreign nuclease into thereaction mixture which attacks the "foreign" target DNA. All cells havesome nonspecific nuclease that attacks foreign DNA that is loose in thecell. When this happens, it interferes with or stops the replicationprocess. Thus, if a drop of saliva or a dandruff particle or materialfrom another sample mixture were inadvertently to enter a samplemixture, the nuclease materials in these cells could attack the targetDNA and cause an error in the amplification process. It is highlydesirable to eliminate all such sources of cross-contamination.

Another source of error is nonprecise control over sample mixturetemperature as between various ones of a multiplicity of differentsamples. For example, if all the samples are not precisely controlled tohave the proper annealing temperature (a user selected temperatureusually in the range from 50° to 60° C.) for the extension incubationcertain forms of DNA will not extend properly. This happens because theprimers used in the extension process anneal to the wrong DNA if thetemperature is too low. If the annealing temperature is too high, theprimers will not anneal to the target DNA at all.

One can easily imagine the consequences of performing the PCRamplification process inaccurately when PCR amplification is part ofdiagnostic testing such as for the presence HIV antibodies, hepatitis,or the presence of genetic diseases such as sickle cell anemia, etc. Afalse positive or false negative result in such diagnostic testing canhave disastrous personal and legal consequences. Accordingly, it is anobject for the design of the PCR instrument described herein toeliminate as many of these sources of possible errors as possible suchas cross-contamination or poor temperature control while providing aninstrument which is compatible with the industry standard 96-wellmicrotiter plate format. The instrument must rapidly perform PCR in aflexible manner with a simple user interface.

In the preferred embodiment, the sample block 12 is machined out of asolid block of relatively pure but corrosion resistant aluminum such asthe 6061 aluminum alloy. Machining the block structure out of a solidblock of aluminum results in a more thermally homogeneous structure.Cast aluminum structures tend not to be as thermally homogenous as isnecessary to meet the very tight desired temperature controlspecifications.

Sample block 12 is capable of rapid changes in temperature because thethermal mass of the block is kept low. This is done by the formation inthe block of many cooling passageways, sample wells, grooves and otherthreaded and unthreaded holes. Some of these holes are used to attachthe block to supports and to attach external devices such as manifoldsand spillage trays thereto.

To best appreciate the "honeycomb" nature of the sample block structure,the reader should refer simultaneously to FIG. 2 which shows the blockin plan view as well as FIGS. 3 through 8 which show elevation views andstrategically located sectional views of the sample block. For example,FIG. 3 is a side elevation view showing the cooling channel positionstaken from the vantage point of the view line 3-3' in FIG. 2. Theelevation view of the sample block 12, looking at the opposite edge, isidentical. FIG. 4 is an elevation view of the edge of the sample block12 from the perspective of view line 4-4' in FIG. 2. FIG. 5 is anelevation view of the end of the sample block 12 taken from theperspective of view line 5-5' in FIG. 2. FIG. 6 is a sectional view ofthe sample block 12 taken along the section line 6-6' in FIG. 2. FIG. 7is a sectional view of the sample block 12 taken along section line 7-7'in FIG. 2. FIG. 8 is a sectional view of the sample block 12 taken alongsection line 8-8' in FIG. 2.

The top surface of the sample block 12 is drilled with an 8×12 array ofconical sample wells of which wells 66 and 68 are typical. The conicalconfiguration of each sample well is best seen if FIG. 8. The walls ofeach sample well are drilled at an angle of 17° to match the angle ofthe conical section of each sample tube. This is done by drilling apilot hole having the diameter D_(w) in FIG. 8. Then a 17° countersinkis used to form the conical walls 67.

The bottom of each sample well includes a sump 70 which has a depthwhich exceeds the depth of penetration of the tip of the sample tube.The sump 70 is created by the pilot hole and provides a small open spacebeneath the sample tube when the sample tube is seated in thecorresponding sample well. This sump provides a space for liquid such ascondensation that forms on the well walls to reside without interferingwith the tight fit of each sample tube to the walls of the sample well.This tight fit is necessary to insure that the thermal conductance fromthe well wall to the sample liquid is uniform and high for each sampletube. Any contamination in a well which causes a loose fit for one tubewill destroy this uniformity of thermal conductance across the array.That is, because liquid is substantially incompressible at the pressuresinvolved in seating the sample tubes in the sample wells, if there wereno sump 70, the presence of liquid in the bottom of the sample wellcould prevent a sample tube from fully seating in its sample well.Furthermore, the sump 70 provides a space in which a gaseous phase ofany liquid residing in the sump 70 can expand during high temperatureincubations such that large forces of such expansion which would bepresent if there were no sump 70 are not applied to the sample tube topush the tube out of flush contact with the sample well.

It has been found experimentally that it is important for each sampletube to be in flush contact with its corresponding sample well and thata certain minimum threshold force be applied to each sample tube to keepthe thermal conductivity between the walls of the sample well and thereaction mixture uniform throughout the array. This minimum thresholdseating force is shown as the force vector F in FIG. 15 and is a keyfactor in preventing the thermal conductivity through the walls of onesample tube from being different than the thermal conductivity throughthe walls of another sample tube located elsewhere in the block. Theminimum threshold seating force F is 30 grams and the preferred forcelevel is between 50 and 100 grams.

The array of sample wells is substantially completely surrounded by agroove 78, best seen in FIGS. 2, 6 and 8, which has two functions. Themain function is to reduce the thermal conductivity from the centralarea of the sample block to the edge of the block. The groove 78 extendsabout 2/3 through the thickness of the sample block. This grooveminimizes the effects of unavoidable thermal gradients caused by thenecessary mechanical connections to the block of the support pins,manifolds, etc. A secondary function is to remove thermal mass from thesample block 12 so as to allow the temperature of the sample block 12 tobe altered more rapidly and to simulate a row of wells in the edgeregion called the "guard band". The amount of metal removed by theportion of the groove 78 between points 80 and 82 in FIG. 2 is designedto be substantially equal to the amount of metal removed by the adjacentcolumn of eight sample wells 83 through 90. The purpose of this is tomatch the thermal mass of the guard band to the thermal mass of theadjacent " local zone", a term which will be explained more fully below.

Referring specifically to FIGS. 3, 6 and 8, there is shown the numberand relative positions of the various bias cooling and ramp coolingchannels which are formed in the metal of the sample block 12. There arenine bias cooling channels marked with reference numerals 91 through 99.Likewise, there are eight ramp cooling channels marked with referencenumerals 100 through 107.

Each of these bias cooling and ramp cooling channels is gun drilledthrough the aluminum of the sample block. The gun drilling process iswell known and provides the ability to drill a long, very straight holewhich is as close as possible to the bottom surface 110 of the sampleblock 12. Since the gun drilling process drills a straight hole, thisprocess is preferred so as to prevent any of the bias cooling or rampcooling channels from straying during the drilling process andpenetrating the bottom surface 110 of the sample block or otherwisealtering its position relative to the other cooling channels. Suchmispositioning could cause undesirable temperature gradients byupsetting the "local balance" and "local symmetry" of the local zones.These concepts are explained below, but for now the reader shouldunderstand that these notions and the structures which implement themare key to achieving rapid temperature cycling of up to 96 sampleswithout creating excessive temperature errors as between differentsample wells.

The bias cooling channels 91 through 99 are lined with silicone rubberin the preferred embodiment to reduce the thermal conductivity acrossthe wall of the bias cooling channel. Lowering of the thermalconductivity across the channel wall in the bias cooling channels ispreferred so as to prevent too rapid of a change in temperature of thesample block 12 when the multizone heater 156 is turned off and heatloss from the sample block 12 is primarily through the bias coolingchannels. This is the situation during the control process carried outwhen the sample block temperature has strayed slightly above the desiredtarget incubation temperature and the control system is attempting tobring the sample block temperature back down to the user's specifiedincubation temperature. Too fast a cooling rate in this situation couldcause overshoot of the desired incubation temperature before the controlsystem's servo feedback loop can respond although a "controlledovershoot" algorithm is used as will be described below. Since the blocktemperature servo feedback loop has a time constant for reacting tostimuli, it is desirable to control the amount of heating and coolingand the resulting rate of temperature change of the sample block suchthat overshoot is minimized by not changing the sample block temperatureat a rate faster than the control system can respond to temperatureerrors.

In the preferred embodiment, the bias cooling channels are 4 millimetersin diameter, and the silicone rubber tube has a one millimeter insidediameter and a 1.5 millimeter wall thickness. This provides a biascooling rate of approximately 0.2° C. per second when the block is atthe high end of the operating range, i.e., near 100° C., and a biascooling rate of approximately 0.1° C. per second when the sample block12 is at a temperature in the lower end of the operating range. Thecoolant control system 24 in FIG. 1 causes a flow rate for coolant inthe bias cooling channels of approximately 1/20th to 1/30th of the flowrate for liquid coolant through the ramp cooling channels, 100 through107. The bias cooling and ramp cooling channels are the same size, i.e.,4 millimeters in diameter, and extend completely through the sampleblock 12.

The bias cooling channels are lined by inserting a stiff wire with ahook at the end thereof through the bias cooling channel and hooking itthrough a hole in the end of a silicone rubber tube which has an outsidediameter which is slightly greater than 4 millimeters. The hook in thewire is then placed through the hole in the silicone rubber tube, andthe silicone tube is pulled through the bias cooling channel and cut offflush with the end surfaces of the sample block 12.

Threaded holes 108 through 114 are used to bolt a coolant manifold toeach side of the sample block 12. There is a coolant manifold bolted toeach end of the block. These two coolant manifolds are coupled to thecoolant channels 26, 28, 30 and 32 in FIG. 1, and are affixed to thesample block 12 with a gasket material (not shown) interposed betweenthe manifold and the sample block metal. This gasket prevents leaks ofcoolant and limits the thermal conductivity between the sample block 12and the manifold which represents a heat sink. Preferably, the gasketmaterial is ethylene propylene. Any gasket material which serves theabove stated purposes will suffice for practicing the invention.

The positions of the bias cooling and ramp cooling channels relative tothe position of the groove 78 are best seen in the sectional view ofFIG. 6. The positions of the bias cooling and ramp cooling channelsrelative to the positions of the sample wells is best seen in FIG. 8.The bias cooling and ramp cooling channels are generally interposedbetween the positions of the tips of the sample wells. Further, FIG. 8reveals that the bias cooling and ramp cooling channels such as channels106 and 97 cannot be moved in the positive z direction very far withoutrisking penetration of the walls of one or more sample wells. Likewise,the cooling channels cannot be moved in the negative z direction veryfar without creating the possibility of penetrating the bottom surface116 of the sample block 12. For clarity, the positions of the bias andramp cooling channels are not shown in hidden lines in FIG. 2 relativeto the positions of the sample wells and other structures. However,there is either a bias cooling channel or a ramp cooling channel betweenevery column of sample wells.

Referring to FIG. 2, the holes 118, 119, 120 and 121 are threaded andare used to attach the sample block 12 to machinery used to machine thevarious holes and grooves formed therein. In FIGS. 2, 4 and 5, the holes124, 125, 126 and 127 are used to attach the sample block 12 to asupport bracket shown in FIG. 9 to be described in more detail below.Steel bolts extend through this support bracket into the threaded holes124 through 127 to provide mechanical support of the sample block 12.These steel bolts also represent heat sinks or heat sources which tendto add thermal mass to the sample block 12 and provide additionalpathways for transfer of thermal energy between the sample block 12 andthe surrounding environment. These support pins and the manifolds aretwo important factors in creating the need for the guard bands toprevent the thermal energy transferred back and forth to theseperipheral structures from affecting these sample temperatures.

Referring to FIG. 5, the holes 128, 130 and 132 are mounting holes foran integrated circuit temperature sensor (not shown) which is insertedinto the sample block through hole 128 and secured thereto by boltswhich fasten to threaded holes 130 and 132. The extent of penetration ofthe hole 128 and the relative position of the temperature sensor to thegroove 78 and the adjacent column of sample wells is best seen in FIG.2.

Referring to FIG. 2, holes 134 through 143 are mounting holes which areused to mount a spill collar 147 (not shown). This spill collar 147 isshown in FIG. 19 detailing the structure of the heated platen 14,sliding cover 316 and lead screw assembly 312. The purpose of the spillcollar is to prevent any liquid spilled from the sample tubes fromgetting inside the instrument casing where it could cause corrosion.

Referring to FIG. 9, there is shown in cross-section a view of thesupport system and multi-zone heater 156 configuration for the sampleblock 12. The sample block 12 is supported by four bolts of which bolt146 is typical. These four bolts pass through upright members of a steelsupport bracket 148. Two large coil springs 150 and 152 are compressedbetween a horizontal portion of the support bracket 148 and a steelpressure plate 154. The springs 150 and 152 are compressed sufficientlyto supply approximately 300 lbs. per square inch of force in thepositive z direction acting to compress a film heater 156 to the bottomsurface 116 of the sample block 12. This three layer film heaterstructure is comprised of a multi-zone film heater 156, a siliconerubber pad 158 and a layer of epoxy resin foam 160. In the preferredembodiment the film heater 156 has three separately controllable zones.The purpose of the film heater 156 is to supply heat to the sample block12 under the control of the CPU 20 in FIG. 1. The purpose of thesilicone rubber pad 158 is to lower the thermal conductivity from thefilm heater layer 156 to the structures below. These lower structuresserve as heat sinks and heat sources between which undesired heat energymay be transferred to and from the sample block 12. The silicone rubberpad 158 has the additional function of compensating for surfaceirregularities in the film heater 156 since some film heaters embodynichrome wires and may not be not perfectly flat.

The purpose of the steel plate 154 and the epoxy resin foam 160 is totransfer the force from the springs 150 and 152 to the silicone rubberpad 158 and the multi-zone film heater 156 so as to compress the filmheater to the bottom surface 116 of the sample block with as flush a fitas possible. The epoxy resin foam should be stiff so as to not becrushed under the force of the springs but it should also be a goodinsulator and should have low thermal mass, i.e., it should be anondense structure. In one embodiment, the foam 160 is manufacturedunder the trademark ECKO foam. In alternative embodiments, otherstructures may be substituted for the silicone rubber layer 158 and/orthe epoxy resin foam layer 160. For example, a stiff honeycomb structuresuch as is used in airplane construction could be placed between thepressure plate 154 and the film heater 156 with insulating layerstherebetween. Whatever structure is used for layers 158 and 160 shouldnot absorb substantial amounts of heat from the sample block 12 whilethe block is being heated and should not transfer substantial amounts ofheat to the sample block 12 when the block is being cooled. Perfectisolation of the block from its surrounding structures however, isvirtually impossible. Every effort should be made in designingalternative structures that will be in contact with the sample block 12so as to thermally isolate the sample block from its environment as muchas possible to minimize the thermal mass of the block and enable rapidtemperature changes of the sample block and the sample mixtures storedtherein.

Precise temperature control of the sample block temperature is achievedby the CPU 20 in FIG. 1 by controlling the amount of heat applied to thesample block by the multi-zone film heater 156 in FIG. 9. The filmheater is driven using a modified form of pulse width modulation. First,the 120 volt waveform from the power line is rectified to preserve onlyhalf cycles of the same polarity. Then portions of each half cycle aregated to the appropriate zones of the foil heater, with the percentageof each half cycle which is applied to the various zones of the foilheater being controlled by the CPU 20.

FIG. 10 illustrates one embodiment of a power control concept for thefilm heater 156. FIG. 10 is a diagram of the voltage waveform of thesupply line voltage. Rectification to eliminate the negative half cycle162 occurs. Only positive half cycles remain of which half cycle 164 istypical. The CPU 20 and its associated peripheral electronic circuitrythen controls the portion of each half cycle which is applied to thevarious zones of the film heater 156 by selecting a portion of each halfcycle to apply according to a power level computed for each zone basedupon equations given below for each zone. That is, the dividing line 166is moved forward or backward along the time axis to control the amountof power to the film heater based upon a number of factors which arerelated in a special equation for each zone. The cross-hatched areaunder the positive half cycle 164 represents the amount of power appliedto the film heater 156 for the illustrated position of the dividing line166. As the dividing line 166 is moved to the right, more power isapplied to the film heater, and the sample block 12 gets hotter. As thedividing line is moved to the left along the time axis, thecross-hatched area becomes smaller and less power is applied to the filmheater. How the CPU 20 and its associated software and peripheralcircuitry control the temperature of block 12 will be described in moredetail below.

The amount of power supplied to the film heater is continuously variablefrom 0 to 600 watts. In alternative embodiments, the amount of powersupplied to the film heater 156 can be controlled using other schemessuch as computer control over the current flow through or voltageapplied to a DC film heater or by the zero crossing switching schemedescribed below.

In other embodiments, heating control of the sample block 12 may beperformed by control over the flow rate and/or temperature of hot gasesor hot liquid which is gated through heating control channels which areformed through the metal of the sample block 12. Of course in suchalternative embodiments, the number of sample wells in the block wouldhave to be reduced since there is no room for additional heatingchannels in the sample block 12 shown in FIGS. 2 through 8. Suchalternative embodiments could still be compatible with the 96-wellmicrotiter plate format if, for example, every other well were removedto make room for a heating channel in the sample block. This wouldprovide compatibility only as to the dimensions of such microtiterplates and not as to the simultaneous processing of 96 differentsamples. Care must be taken to preserve local balance and local symmetryin these alternative embodiments.

In the embodiment described herein, the maximum power that can bedelivered to the block via the film heater is 1100 watts. Thislimitation arises from the thermal conductivity of the block/heaterinterface. It has been found experimentally that the supply of more thanapproximately 1100 watts to the film heater 156 will frequently causeself-destruction of the device.

Typical power for heating or cooling when controlling block temperaturesat or near target incubation temperatures is in the range of plus orminus 50 watts.

Referring to FIG. 11, there is shown a time versus temperature plot of atypical PCR protocol. Large downward changes in block temperature areaccomplished by gating chilled liquid coolant through the ramp coolingchannels while monitoring the sample block temperature by thetemperature sensor 21 in FIG. 1. Typically these rapid downwardtemperature changes are carried out during the ramp following thedenaturation incubation 170 to the temperature of hybridizationincubation 172. Typically, the user must specify the protocol bydefining the temperatures and times in one fashion or another so as todescribe to the CPU 20 the positions on the temperature/time plane ofthe checkpoints symbolized by the circled intersections between the ramplegs and the incubation legs. Generally, the incubation legs are markedwith reference numerals 170, 172 and 174 and the ramps are marked withreference numerals 176, 178 and 180. Generally the incubation intervalsare conducted at a single temperature, but in alternative embodiments,they may be stepped or continuously ramped to different temperatureswithin a range of temperatures which is acceptable for performing theparticular portion of the PCR cycle involved. That is, the denaturationincubation 170 need not be carried out at one temperature as shown inFIG. 11, but may be carried out at any of a plurality of differenttemperatures within the range of temperatures acceptable fordenaturation. In some embodiments, the user may specify the length ofthe ramp segments 176, 178 and 180. In other embodiments, the user mayonly specify the temperature or temperatures and duration of eachincubation interval, and the instrument will then move the temperatureof the sample block as rapidly as possible between incubationtemperatures upon the completion of one incubation and the start ofanother. In the preferred embodiment, the user can also havetemperatures and/or incubation times which are different for each cycleor which automatically increment on every cycle.

The average power of ramp cooling during a transition from a 95° C.denaturation incubation to a 35° C. hybridization incubation is morethan one kilowatt typically. This results in a temperature change forthe sample block of approximately 4°-6° C. per second when the blocktemperature is at the high end of the operating range, and approximately2° C. per second when the block temperature is at the low end of theoperating range. Generally it is desirable to have as high a coolingrate as possible for ramp cooling.

Because so much heat is being removed from the sample block during rampcooling, temperature gradients across the sample block from one end of aramp cooling channel to the other could occur. To prevent this andminimize these types of temperature gradients, the ramp cooling channelsare directionally interlaced. That is, in FIG. 3, the direction ofcoolant flow through ramp cooling channels 100, 102, 104, and 106 isinto the page as symbolized by the x's inside these ramp cooling channelholes. Ramp cooling liquid flow in interlaced ramp cooling channels 101,103, 105, and 107 is out of the page as symbolized by the single pointsin the center of these ramp cooling channel holes. This interlacing plusthe high flow rate through the ramp cooling channels minimizes anytemperature gradients which might otherwise occur using noninterlacedflow patterns or lower flow rates because the distances between the hotand cold ends of the channels is made smaller. A slower flow rateresults in most or all of the heat being taken from the block in thefirst inch or so of travel which means that the input side of the blockwill be at a lower temperature than the output side of the block. A highflow rate minimizes the temperature gradient along the channel.Interlacing means the hot end of the channels running in one directionare "sandwiched" between the cold ends of channels wherein flow is inthe opposite direction. This is a smaller distance than the length ofthe channel. Thus, temperature gradients are reduced because thedistances heat must travel to eliminate the temperature gradient arereduced. This causes any temperature gradients that form because ofcooling in the ramp channels to be quickly eliminated before they havetime to differentially heat some samples and not others. Withoutinterlacing, one side of the sample block would be approximately 1° C.hotter than the other side. Interlacing results in dissipation of anytemperature gradients that result in less than approximately 15 seconds.

In order to accurately estimate the amount heat added to or removed fromthe block, the CPU 20 measures the block temperature using temperaturesensor 21 in FIG. 1 and measures the coolant temperature by way oftemperature sensor 61 in FIG. 46 coupled to bus 54 in FIG. 1. Theambient air temperature is also measured by way of temperature sensor 56in FIG. 1, and the power line voltage, which controls the power appliedto the film heaters on bus 52, is also measured. The thermal conductancefrom the sample block to ambient and from the sample block to thecoolant are known to the CPU 20 as a result of measurements made duringan initialization process to set control parameters of the system.

For good temperature uniformity of the sample population, the block, atconstant temperature, can have no net heat flow in or out. However,temperature gradients can occur within the sample block arising fromlocal flows of heat from hot spots to cold spots which have zero netheat transfer relative to the block borders. For instance, a slab ofmaterial which is heated at one end and cooled at the other is at aconstant average temperature if the net heat flow into the block iszero. However, in this situation a significant temperaturenonuniformity, i.e., a temperature gradient, can be established withinthe slab due to the flow of heat from the hot edge to the cold edge.When heating and cooling of the edges of the block are stopped, the flowof heat from the hot edge to the cold edge eventually dissipates thistemperature gradient and the block reaches a uniform temperaturethroughout which is the average between the hot temperature and cooltemperature at the beginning of heat flow.

If a slab of cross sectional area A in length L has a uniform thermalconductivity K, and the slab is held at constant average temperaturebecause heat influx from a heat source Q_(in) is matched by heat outflowto a heat sink Q_(out), the steady state temperature profile whichresults from the heat flow is: ##EQU1## Where, Delta T=the temperaturegradient

L=the thermal path length

A=the area of the thermal path

K=the thermal conductance through the path

In general, within any material of uniform thermal conductance, thetemperature gradient will be established in proportion to the heat flowper unit area. Heat flow and temperature nonuniformity are thusintimately linked.

Practically speaking, it is not possible to control the temperature of asample block without some heat flow in and out. The cold bias controlcooling requires some heat flow in from the strip heaters to balance theheat removed by the coolant flowing through the bias cooling channels tomaintain the block temperature at a stable value. The key to a uniformsample block temperature under these conditions is a geometry which has"local balance" and "local symmetry" of heat sources and heat sinks bothstatically and dynamically, and which is arranged such that any heatflow from hot spots to cold spots occurs only over a short distance.

Stated briefly, the concept of "static local balance" means that in ablock at constant temperature where the total heat input equals thetotal heat output, the heat sources and heat sinks are arranged suchthat within a distinct local region, all heat sources are completelybalanced by heat sinks in terms of heat flows in and heat flows out ofthe block. Therefore, each local region, if isolated, would bemaintained at a constant temperature.

The concept of "static local symmetry" means that, within a local regionand for a constant temperature, the center of mass of heat sources iscoincident with the center of mass of heat sinks. If this were not thecase, within each local region, a temperature gradient across each localregion can exist which can add to a temperature gradient in an adjacentlocal region thereby causing a gradient across the sample block which istwice as large as the size of a single local region because of lack oflocal symmetry even though local balance within each local regionexists. The concepts of local balance and local symmetry are importantto the achievement of a static temperature balance where the temperatureof the sample block is being maintained at a constant level during, forexample, an incubation interval.

For the dynamic case where rapid temperature changes in the sample blockare occurring, the thermal mass, or heat capacity of each local regionbecomes important. This is because the amount of heat that must flowinto each local region to change its temperature is proportional to thethermal mass of that region.

Therefore, the concept of static local balance can be expanded to thedynamic case by requiring that if a local region includes x percent ofthe total dynamic heat source and heat sink, it must also include xpercent of the thermal mass for "dynamic local balance" to exist.Likewise, "dynamic local symmetry" requires that the center of mass ofheat capacity be coincident with the center of mass of dynamic heatsources and sinks. What this means in simple terms is that the thermalmass of the sample block is the metal thereof, and the machining of thesample block must be symmetrical and balanced such that the total massof metal within each local zone is the same. Further, the center of massof the metal in each local zone should be coincident with the center ofmass of the dynamic heat sources and sinks. Thus, the center of mass ofthe multi-zone heater 156, i.e., its geometric center, and the geometriccenter of the bias and ramp cooling channels must coincide. From a studyof FIGS. 2-9, it will be seen from the detailed discussion below thatboth static and dynamic local balance and local symmetry exist in sampleblock 12.

FIG. 12 illustrates two local regions side by side for the design of thesample block 12 according to the teachings of the invention. In FIG. 12,the boundaries of two local regions, 200 and 202, are marked by dashedlines 204, 206 and 208. FIG. 12 shows that each local region which isnot in the guard band is comprised of: two columns of sample wells; aportion of the foil heater 156 which turns out to be 1/8th of the totalarea of the heater; one ramp cooling channel such as ramp coolingchannels 210 and 212; and, one bias cooling channel. To preserve localsymmetry, each local region is centered on its ramp cooling channel andhas one-half of a bias cooling channel at each boundary. For example,local region 200 has a center over the ramp cooling channel 210 and biascooling channels 214 and 216 are dissected by the local regionboundaries 204 and 206, respectively. Thus the center of mass of theramp cooling channel (the middle thereof), coincides (horizontally) withthe center of mass of the bias cooling channels (the center of the localregion) and with the center of mass of the film heater portion coupledto each local region. Static local balance will exist in each localregion when the CPU 20 is driving the film heater 156 to input an amountof heat energy that is equal to the amount of heat energy that is beingremoved by the ramp cooling and bias cooling channels. Dynamic localbalance for each local region exists because each local region in thecenter portion of the block where the 96 sample mixtures reside containsapproximately 1/8th the total thermal mass of the entire sample block,contains 1/8th of the total number of ramp cooling channels and contains1/8th of the total number of bias cooling channels. Dynamic localsymmetry exists for each local region, because the center of mass of themetal of each local region is horizontally coincident with: the centerof film heater portion underlying the local region; the center of theramp cooling channel; and, the center of mass of the two half biascooling channels.

By virtue of these physical properties characterized as static anddynamic local balance and local symmetry, the sample block heats andcools all samples in the population much more uniformly than prior artthermal cyclers.

Referring to FIG. 2, the plan view of the boundaries of the localregions are illustrated by dashed lines 217 through 225. Inspection ofFIG. 2 reveals that the central region of the 96 sample wells aredivided into six adjacent local regions bounded by boundaries 218through 224. In addition, two guard band local regions are added at eachedge. The edge local region (local regions are sometimes herein alsocalled local zones) having the most negative x coordinate is bounded byboundary lines 217 and 218. The edge local region having the mostpositive x coordinate is bounded by boundary lines 224 and 225. Notethat the edge local regions contain no sample well columns but docontain the groove 78 simulating a column of wells. The depth and widthof the groove 78 is designed to remove the same metal mass as a columnof wells thereby somewhat preserving dynamic local symmetry. The edgelocal zones are therefore different in thermal mass (they also haveadditional thermal mass by virtue of the external connections such asmanifolds and support pins) than the six local zones in the central partof the sample block. This difference is accounted for by heating theedge local zones or guard bands with separately controllable zones ofsaid multizone heater so that more energy may be put into the guard bandthan the central zone of the block.

The local regions at each edge of the block approximate, but do notexactly match the thermal properties of the six centrally located localregions. The edge local regions are called "guard band" regions becausethey complete a guard band which runs around the periphery of the sampleblock 12. The purpose of this guard band is to provide some thermalisolation of the central portion of the sample block containing the 96sample wells from uncontrolled heat sinks and sources inherentlyembodied in mechanical connections to the block by such things assupport pins, manifolds, drip collars and other devices which must bemechanically affixed to the sample block 12. For example in FIG. 2, theedge surfaces 228 and 230 of the sample block have plastic manifoldsattached thereto which carry coolant to and from the ramp and biascooling passages. The guard band along edges 228 and 230 consists ofportions of the slot 78 which are parallel to and closest to the edges228 and 230. The depth of the groove 78 is such that the bottom of thegroove is as close to the perimeters of the bias and ramp coolingchannels as is possible without actually intersecting them. The width ofthe groove 78 coupled with this depth is such that the volume of metalremoved by the slot 78 between points 82 and 232 in FIG. 2 approximatelyequals the volume of metal removed by the adjacent row of sample wellsstarting with sample well 234 and ending with sample well 83. Also, theslot 78 all around the perimeter of the block is located approximatelywhere such an additional row of wells would be if the periodic patternof sample wells were extended by one row or column of wells in eachdirection.

Along the edges 250 and 252 where the support connections are made tothe sample block, the guard band local regions contain, in addition to aportion of the slot 78, the full length of several cooling channels.Referring to FIG. 3, these include: 1/2 of a bias cooling channel (e.g.,92) which merges with the adjacent 1/2 bias cooling channel of theadjacent local region to form a whole bias cooling channel; a rampcooling channel (e.g., 100); and a whole bias cooling channel (e.g.,91). For the edge local region at edge 250, these cooling channels are107, 198 and 99.

The whole bias cooling channels in the guard bands are slightlydisplaced inward from the edge of the block. The reason that these wholebias cooling channels are used is because a "half" cooling channel isimpractical to build. Since the bias cooling channels require such athick walled rubber lining, it would be difficult to keep a hole througha lining of a "half" bias cooling channel reliably open. This asymmetryin the edge local regions causes a small excess loss of heat to thecoolant from the edge guard band local regions, but it is sufficientlyremote from the central region of the sample block containing the samplewells that its contribution to sample temperature nonuniformities issmall. Also, since the temperature affects of this small asymmetry arepredictable, the effect can be further minimized by the use of aseparately controllable zone of the multi-zone heater system under eachguard band.

Referring to FIG. 13, there are shown three separately controlled zoneswithin the film heater layer 156 in FIG. 9. These separately controlledzones include edge heater zones which are situated under the guard bandsat the exposed edges of the sample block 12 which are coupled to thesupport bracket 148. There are also separately controlled manifoldheater zones situated under the guard bands for the edges 228 and 230which are attached to the coolant manifolds. Finally, there is a centralheater zone that underlies the sample wells. The power applied to eachof these zones is separately controlled by the CPU 20 and the controlsoftware.

The film heater 156 is composed of a pattern of electrical conductorsformed by etching a thin sheet of metal alloy such as Inconel™. Themetal alloy selected should have high electrical resistance and goodresistance to heat. The pattern of conductors so etched is bondedbetween thin sheets of an electrically insulating polymeric materialsuch as Kapton™. Whatever material is used to insulate the electricalresistance heating element, the material must be resistant to hightemperatures, have a high dielectric strength and good mechanicalstability.

The central zone 254 of the film heater has approximately the samedimensions as the central portion of the sample block inside the guardbands. Central region 254 delivers a uniform power density to the samplewell area.

Edge heater regions 256 and 258 are about as wide as the edge guardbands but are not quite as long.

Manifold heater regions 260 and 262 underlie the guard bands for edges228 and 230 in FIG. 2.

The manifold heater zones 260 and 262 are electrically connectedtogether to form one separately controllable heater zone. Also, the edgeheater sections 256 and 258 are electrically coupled together to form asecond separately controllable heater zone. The third separatelycontrollable heater zone is the central section 254. Each of these threeseparately controllable heater zones has separate electrical leads, andeach zone is controlled by a separate control algorithm which may be runon separate microprocessors or a shared CPU as is done in the preferredembodiment.

The edge heater zones 256 and 258 are driven to compensate for heat lostto the support brackets. This heat loss is proportional to thetemperature difference between the sample block 12 and the ambient airsurrounding it. The edge heater zones 256 and 258 also compensate forthe excess loss of heat from the sample block to the full bias coolingchannels at each edge of the block. This heat loss is proportional tothe temperature difference between the sample block 12 and the coolantflowing through these bias cooling channels.

The manifold heater sections 260 and 262 are also driven so as tocompensate for heat lost to the plastic coolant manifolds 266 and 268 inFIG. 13 which are attached to the edges of the sample block 12. Thepower for the manifold heater sections 260 and 262 compensates for heatloss which is proportional mainly to the temperature difference betweenthe sample block and the coolant, and to a lesser degree, between thesample block and the ambient air.

For practical reasons, it is not possible to match the thermal mass ofthe guard band local regions with the thermal masses of the localregions which include the sample wells overlying central heater section254. For example, the plastic coolant manifolds 266 and 268 not onlyconduct heat away from the guard band, but they also add a certainamount of thermal mass to the guard band local regions to which they areattached. The result of this is that during rapid block temperaturechanges, the rates of rise and fall of guard band temperature do notexactly match that of the sample well local regions. This generates adynamic temperature gradient between the guard bands and sample wells,which if allowed to become large, could persist for a time which islonger than is tolerable. This temperature gradient effect is roughlyproportional to the rate of change of block temperature and is minimizedby adding or deleting heat from each guard band local zone at a ratewhich is proportional to the rate of change of block temperature.

The coefficients of proportionality for the guard band zone heaters arerelatively stable properties of the design of the system, and aredetermined by engineering measurements on prototypes. The values forthese coefficients of proportionality are given below in connection withthe definitions of the terms of Equations (3) through (5). Theseequations define the amounts of power to be applied to the manifoldheater zone, the edge heater zone and the central zone, respectively inan alternative embodiment. The equations used in the preferredembodiment are given below in the description of the software (Equations(46)-(48), power distributed by area).

    P.sub.m =A.sub.m P+K.sub.ml (T.sub.BLK -T.sub.AMB)+K.sub.M2 (T.sub.BLK -T.sub.COOL)+K.sub.M3 (dt.sub.BLK /dt)                    (3)

where,

P_(m) =power supplied to the manifold heater zones 260 and 262.

A_(m) =area of the manifold heater zone.

P=power needed to cause the block temperature to stay at or move to thedesired temperature at any particular time in a PCR thermal cycleprotocol.

K_(M1) =an experimentally determined constant of proportionality tocompensate for excess heat loss to ambient through the manifolds, equalto 0 watts/degree Kelvin.

K_(M2) =an experimentally determined constant of proportionality tocompensate for excess heat loss to the coolant, equal to 0.4watts/degree Kelvin.

K_(M3) =an experimentally determined constant of proportionality toprovide extra power to compensate for additional thermal mass of themanifold edge guard bands caused by the attachment of the plasticmanifolds etc., equal to 66.6 watt-seconds/degree Kelvin.

T_(BLK) =the temperature of the sample block 12.

T_(AMB) =the temperature of the ambient air.

T_(COOL) =the temperature of the coolant.

dt_(BLK) /dt=the change in sample block temperature per unit time.

    P.sub.E =A.sub.E P+K.sub.E1 (T.sub.BLK -T.sub.AMB)+K.sub.E2 (T.sub.BLK -T.sub.COOL)+K.sub.E3 (dt.sub.BLK /dt)                    (4)

where,

P_(E) =power to be applied to the edge heater zones

A_(E) =the area of the edge heater zones

K_(E1) =an experimentally determined constant of proportionality tocompensate for excess heat loss to ambient through the manifolds, equalto 0.5 watts/degree Kelvin.

K_(E2) =an experimentally determined constant of proportionality tocompensate for excess heat loss to the coolant, equal to 0.15watts/degree Kelvin.

K_(E3) =an experimentally determined constant of proportionality toprovide extra power to compensate for additional thermal mass of theexposed edge guard bands caused by the attachment of the sample block 12to the support pins and bracket, the temperature sensor etc., equal to15.4 watt-sec/degree Kelvin.

    P.sub.c =A.sub.c P                                         (5)

where

P_(c) =the power to be applied to the central zone 254 of the multi-zoneheater.

A_(c) =the area of the central zone 254.

In each of Equations (3) through (5), the power term, P is a variablewhich is calculated by the portion of the control algorithm run by theCPU 20 in FIG. 1 which reads the user defined setpoints and determineswhat to do next to cause the sample block temperature to stay at orbecome the proper temperature to implement the PCR temperature protocoldefined by the time and temperature setpoints stored in memory by theuser. The manner in which the setpoints are read and the power densityis calculated will be described in more detail below.

The control algorithm run by CPU 20 of FIG. 1 senses the temperature ofthe sample block via temperature sensor 21 in FIG. 1 and FIG. 9 and bus52 in FIG. 1. This temperature is differentiated to derive the rate ofchange of temperature of the sample block 12. The CPU then measures thetemperature of the ambient air via temperature sensor 56 in FIG. 1 andmeasures the temperature of the coolant via the temperature sensor 61 inthe coolant control system 24 shown in FIG. 46. The CPU 20 then computesthe power factor corresponding to the particular segment of the PCRprotocol being implemented and makes three calculations in accordancewith Equations (3), (4) and (5) by plugging in all the measuredtemperatures, the constants of proportionality (which are stored innonvolatile memory), the power factor P for that particular iteration ofthe control program and the areas of the various heater zones (which arestored in nonvolatile memory). The power factor is the total powerneeded to move the block temperature from its current level to thetemperature level specified by the user via a setpoint. More details onthe calculations performed by the CPU to control heating and cooling aregiven below in the description of the control software "PID task".

After the required power to be applied to each of the three zones of theheater 156 is calculated, another calculation is made regarding theproportion of each half cycle of input power which is to be applied toeach zone in some embodiments. In the preferred embodiment describedbelow, the calculation mode is how many half cycles of the total numberof half cycles which occur during a 200 millisecond sample period are tobe applied to each zone. This process is described below in connectionwith the discussion of FIGS. 47A and 47B (hereafter referred to as FIG.47) and the "PID Task" of the control software. In the alternativeembodiment symbolized by FIG. 10, the computer calculates for each zone,the position of the dividing line 166 in FIG. 10. After this calculationis performed, appropriate control signals are generated to cause thepower supplies for the multi-zone heater 156 to do the appropriateswitching to cause the calculated amount of power for each zone to beapplied thereto.

In alternative embodiments, the multi-zone heater can be implementedusing a single film heater which delivers uniform power density to theentire sample block, plus one or two additional film heaters with onlyone zone apiece for the guard bands. These additional heaters aresuperimposed over the single film heater that covers the entire sampleblock. In such an embodiment, only the power necessary to make up theguard band losses is delivered to the additional heater zones.

The power factor P in Equations (3) through (5) is calculated by the CPU20 for various points on the PCR temperature protocol based upon the setpoints and ramp times specified by the user. However, a limitation isimposed based upon the maximum power delivery capability of the zoneheater mentioned above.

The constants of proportionality in Equations (3) through (5) must beproperly set to adequately compensate for excess heat losses in theguard band for good temperature uniformity.

Referring to FIG. 17, there is shown a graph of the differences betweencalculated sample temperatures for a plurality of different sample inresponse to a step change in block temperature to raise the temperatureof the sample block toward a denaturation incubation target temperatureof approximately 94° C. from a substantially lower temperature. FIG. 17illustrates the calculated sample liquid temperatures when themulti-zone heater 156 is properly managed using the constants ofproportionality given above in the definitions of the terms forEquations (3) through (5). The various wells which were used to derivethe graph of FIG. 17 are indicated thereon by a single letter and numbercombination. The 8×12 well array showing FIG. 2 is coded by letteredcolumns and numbered rows. Thus, for example, sample well 90 is alsodesignated sample well A12, while sample well 89 is also designatedsample well B12. Likewise, sample well 68 is also designated sample wellD6, and so on. Note that the well temperatures settle in asymptoticallyat temperatures which are within approximately 0.5° C. of each otherbecause of the overall thermal design described herein to eliminatetemperature gradients.

The foregoing description illustrates how the sample block temperaturemay be controlled to be uniform and to be quickly changeable. However,in the PCR process, it is the temperature of the sample reaction mixtureand not the block temperature that is to be programmed. In the preferredembodiment according to the teachings of the invention, the userspecifies a sequence of target temperatures for the sample liquid itselfand specifies the incubation times for the sample liquid at each ofthese target temperatures for each stage in the PCR process. The CPU 20then manages the sample block temperature so as to get the samplereaction mixtures to the specified target incubation temperatures and tohold the sample mixtures at these target temperatures for the specifiedincubation times. The user interface code run by the CPU 20 displays, atall stages of this process, the current calculated sample liquidtemperature on the display of terminal 16.

The difficulty with displaying an actual measured sample temperature isthat to measure the actual temperature of the reaction mixture requiresinsertion of a temperature measuring probe therein. The thermal mass ofthe probe can significantly alter the temperature of any well in whichit is placed since the sample reaction mixture in any particular well isoften only 100 microliters in volume. Thus, the mere insertion of atemperature probe into a reaction mixture can cause a temperaturegradient to exist between that reaction mixture and neighboringmixtures. Since the extra thermal mass of the temperature sensor wouldcause the reaction mixture in which it is immersed to lag behind intemperature from the temperatures of the reaction mixtures in otherwells that have less thermal mass, errors can result in theamplification simply by attempting to measure the temperature.

Accordingly, the instrument described herein calculates the sampletemperature from known factors such as the block temperature history andthe thermal time constant of the system and displays this sampletemperature on the display. It has been found experimentally for thesystem described herein that if the sample tubes are pressed down intothe sample wells with at least a minimum threshold force F, then for thesize and shape of the sample tubes used in the preferred embodiment andthe sample volumes of approximately 100 microliters, thermally drivenconvection occurs within the sample reaction mixture and the system actsthermally like a single time constant, linear system. Experiments haveshown that each sample tube must be pushed down with approximately 50grams of force for good well-wall-to-liquid thermal conductivity fromwell to well. The heated platen design described below is designed topush down on each sample tube with about 100 grams of force. Thisminimum force, symbolized by force vector F in FIG. 15, is necessary toinsure that regardless of slight differences in external dimensions asbetween various sample tubes and various sample wells in the sampleblock, they all will be pushed down with sufficient force to guaranteethe snug and flush fit for each tube to guarantee uniform thermalconductivity. Any design which has some sample tubes with loose fits intheir corresponding sample wells and some tubes with tight fits will notbe able to achieve tight temperature control for all tubes because ofnon-uniform thermal conductivity. An insufficient level of force Fresults in a temperature response of the sample liquid to a step changein block temperature as shown at 286 in FIG. 14. An adequate level offorce F results in the temperature response shown at 282.

The result achieved by the apparatus constructed according to theteachings of the invention is that the temperature of each samplemixture behaves as if the sample is being well mixed physically duringtransitions to new temperatures. In fact, because of the convectioncurrents caused in each sample mixture, the sample reaction mixture ineach sample tube is being well mixed.

The surprising result is that the thermal behavior of the entire systemis like an electrical RC circuit with a single time constant of 9seconds which is about 1.44 times the half-life of the decay of thedifference between the block temperature and the sample temperature. AGeneAmp™ sample tube filled with 50 milliliters of sample has a timeconstant of about 23 seconds. In other words, during an upward change intemperature of the sample block, the temperature of the reaction mixtureacts like the rise in voltage on the capacitor C in a series RCelectrical circuit like that shown in FIG. 16D in response to a stepchange in the voltage output of the voltage source V.

To illustrate these concepts, refer to FIG. 14 which shows differenttemperature responses of the sample liquid to a step change in blocktemperature and to FIG. 15 which shows a cross section through a samplewell/sample tube combination. It has been found experimentally that whenthe volume of sample liquid 276 is approximately 100 microliters and thedimensions of the tube are such that the meniscus 278 is located belowthe top surface 280 of the sample block 12, and the force F pushing thesample tube into the sample well is at least 30 grams, the thermal timeconstant τ (tau) of the system shown in FIG. 15 is approximately nineseconds for a sample tube wall thickness in the conical section of 0.009inches (dimension A). It has also been found experimentally that forthese conditions, the thermal time constant τ varies by about 1 secondfor every 0.001 inch change in wall thickness for the sample tubefrustum (cone). Thicker tube walls result in longer time constants andmore lag between a change in sample block temperature and the resultingchange in sample liquid temperature.

Mathematically, the expression for the thermal response of the sampleliquid temperature to a change in temperature of the sample block is:

    T.sub.sample =ΔT(1--e.sup.-t/τ)                  (6)

where

T_(sample) =temperature of the sample liquid

ΔT=the temperature difference between the temperature of the sampleblock 12 and the temperature of the sample liquid

t=elapsed time

τ=thermal time constant of the system, or the heat capacity of sampledivided by the thermal conductance from sample well wall to the sampleliquid

In FIG. 14, the curve 282 represents this exponential temperatureresponse to a theoretical step change in sample block temperature whenthe force F pushing down on the sample tube is sufficiently high. Thestep change in temperature of the sample block is shown as function 284,with rapid rise in temperature starting at time T₁. Note how thetemperature of the sample liquid exponentially increases in response tothe step change and asymptotically approaches the final sample blocktemperature. As mentioned briefly above, the curve 286 represents thethermal response when the downward seating force F in FIG. 15 isinsufficient to cause a snug, flush fit between the cone of the sampletube and the wall 290 of the sample well. Generally, the thermalresponse of curve 286 will result if the force F is less than 30 grams.Note that although FIG. 15 shows a small layer of air between the coneof the sample tube and the sample well wall for clarity, this is exactlythe opposite of the desired situation since air is a good insulator andwould substantially increase the thermal time constant of the system.

The thermal time constant τ is analogous to the RC time constant in aseries RC circuit where R corresponds to the thermal resistance betweenthe wall of the sample well and the sample liquid and C is the heatcapacity of the sample liquid. Thermal resistance is equal to theinverse of thermal conductance which is expressed in units watts-secondsper degree Kelvin.

Because of the convection currents 292 shown in the sample liquid inFIG. 15, everywhere in the reaction mixture the sample liquid is at verynearly the same temperature, and the flow of heat between the block andthe sample is very nearly proportional to the difference in temperaturebetween the sample block and the sample reaction mixture. The constantof proportionality is the thermal conductance between the wall of thesample well in the sample block 12 and the reaction mixture. Fordifferent sample volumes or different tubes, i.e., different wallthicknesses or materials, the thermal time constant will be different.In such a case, the user can as part of his specification of the PCRprotocol enter the sample volume or tube type and the machine willautomatically look up the correct thermal time constant for use incalculating the sample temperature. In some embodiments, the user mayenter the actual time constant, and the machine will use it for sampletemperature temperature calculation.

To keep the thermal time constant as small as possible, the conicalwalls of the sample tubes should be as thin as possible. In oneembodiment, these conical walls are 0.009 inches thick whereas the wallsof the cylindrical portion of the sample tube are 0.030 inches thick.The conical shape of the sample tube provides a relatively large surfacearea of contact with the metal of the sample well wall in relation tothe volume of the sample mixture. The tube-to-tube variation of the sizeand shape of the conical section should be controlled so that variationin projection of the tube above the block when the tube is seated in thesample well is within a range of 0.010 inches.

Molding of the sample tubes is done using a "cold runner" system and afour cavity mold such that four sample tubes are molded at eachinjection. The molten plastic is injected at the tip of the sample tubecone so that any remnant of plastic will project into the cavity 291between the tip of the sample tube and the tip of the sample well. Thisprevents any remnant from interfering with the flush fit between thetube and the well. A maximum limit of 0.030 inches is placed on the sizeof any remnant plastic.

In various embodiments, 3 different grades of polypropylene each withdifferent advantages can be used. The preferred polypropylene is PD701from Himont because it is autoclavable. However this plastic isdifficult to mold because it has a low melt index. This plastic has amelt index of 35 and a molecular density of 9. PD701 tends to leaveflash and creates somewhat spotty quality parts but would work better ifit was injected into the thick walled part of the mold instead of at thetip of the conical section as is currently done. Generally, it isdesirable to have a high melt index for ease of molding but also a highmolecular density to maintain good strength and to prevent crazing orcracks under the thermal stress of the autoclaving process at 260° F.Another plastic, PPW 1780 from American Hoescht has a melt index of 75and a molecular density of 9 and is autoclavable. Another plastic whichmay be used in some embodiments is Himont 444. This plastic is notautoclavable and needs to be sterilized in another manner.

In alternative embodiments, the tubes may be molded using a "hotrunner"or "hot nozzle" system where the temperature of the moltenplastic is controlled right up to the gate of the mold. Also, in someembodiments, multiple gates may be used. However, neither of thesetechniques has been experimentally proven at the time of filing to bebetter than the currently used "cold runner" system.

The fact that the system acts thermally like a single time constant RCcircuit is an important result, because it means that if the thermalconductance from the sample block to the sample reaction mixture isknown and uniform, the thermal response of the sample mixtures will beknown and uniform. Since the heat capacity of the sample reactionmixture is known and constant, the temperature of the sample reactionmixture can be computed accurately using only the measured history ofthe block temperature over time. This eliminates the need to measure thesample temperature thereby eliminating the errors and mechanicaldifficulty of putting a probe with nonnegligible thermal mass into asample well to measure the sample temperature directly thereby changingthe thermal mass of the sample in the probed well.

The algorithm which makes this calculation models the thermal behaviorof the system after a single time constant series R-C electricalcircuit. This model uses the ratio of the heat capacity of the liquidsample divided by the thermal conductance from the sample block to thesample reaction mixture. The heat capacity of the sample reactionmixture is equal to the specific heat of the liquid times the mass ofthe liquid. The thermal resistance is equal to one over the thermalconductance from the sample block to the liquid reaction mixture throughthe sample tube walls. When this ratio of heat capacity divided bythermal conductance is expressed in consistent units, it has thedimension of time. For a fixed sample volume and a fixed samplecomposition both of which are the same in every sample well and a fixedthermal conductance, the ratio is also a constant for every sample well,and is called the thermal time constant of the system. It is the timerequired for the sample temperature to come within 36.8% of the blocktemperature after a sudden step change in the block temperature.

There is a mathematical theorem used in the analysis of electroniccircuits that holds that it is possible to calculate the output responseof a filter or other linear system if one knows the impulse response ofthe system. This impulse response is also known as the transferfunction. In the case of a series RC circuit, the impulse response is anexponential function as shown in FIG. 16A. The impulse stimulusresulting in the response of FIG. 16A is as shown in FIG. 16B. Themathematical theorem referred to above holds that the output response ofsuch a linear system can be determined by calculating the convolution ofthe input signal and a weighting function where the weighting functionis the impulse response of the system reversed in time. The convolutionis otherwise known as a running weighted average although a convolutionis a concept in calculus with infinitely small step sizes whereas arunning weighted average has discreet step sizes, i.e., multiplesamples. The impulse response of the series RC circuit shown in FIG. 16Das such that when the voltage of the voltage generator V suddenly risesand falls with a spike of voltage as shown in FIG. 16B, the voltage onthe capacitor C suddenly rises to a peak at 294 in FIG. 16A which isequal to the peak voltage of the impulse shown in FIG. 16B and thenexponentially decays back to the steady state voltage V₁. The resultingweighting function is the impulse response of FIG. 16A turned around intime as shown in FIG. 16C at 385.

Superimposed upon FIG. 16C is a hypothetical curve 387 illustrating atypical temperature history for the temperature of the sample block 12for an approximate step change in temperature. Also shown superimposedupon FIG. 16C are the times of five temperature sample periods labelledT₁ through T₅. According to the teachings of the invention, the sampletemperature is calculated by multiplying the temperature at each one ofthese times T₁ through T₅ by the value of the weighting function at thatparticular time and then summing all these products and dividing by 5.The fact that the thermal system acts like a single time constant linearcircuit is a surprising result based upon the complexities of thermalheat transfer considerations for this complicated thermal system.

In one embodiment, the calculation of the sample temperature is adjustedby a short delay to account for transport lag caused by differentthermal path lengths to the block temperature sensor and the sampleliquid. The calculated sample temperature is displayed for the user'sinformation on the terminal 16 shown in FIG. 1.

FIG. 17 shows the temperature response results for six different wellsspread throughout the 96 well sample block for a step change in sampleblock temperature from a relatively lower temperature in thehybridization/extension temperature range to the relatively highertemperature of approximately 94° C. used for denaturation. The graph ofFIG. 17 shows good agreement between the predicted exponential rise insample temperature if the system were perfectly analogous to the seriesRC circuit shown in FIG. 16D, and also shows excellent uniformity oftemperature response in that the temperatures of the six sample wellsused for this study asymptotically settle in at temperatures very closeto each other and in a denaturation temperature "tolerance" band whichis approximately 0.5° C. wide.

In one embodiment, the ten most recent block temperature samples areused for the running weighted average, but in other embodiments adifferent number of temperature history samples may be used. The goodagreement with theoretically predicted results stems from the fact thatthe thermal convection currents make the sample liquids well mixedthereby causing the system to act in a linear fashion.

The uniformity between sample temperatures in various sample wellsspread throughout the 96 well array results from dynamic and staticlocal balance and local symmetry in the sample block structure as wellas all the other thermal design factors detailed herein. Note howeverthat during rapid temperature changes all the sample wells will havetemperatures within 0.5° C. of each other only if the user has carefullyloaded each sample well with the same mass of sample liquid. Inequalityof mass in different wells does not cause unequal temperatures in steadystate, unchanging conditions, only during rapid changes. The mass of thesample liquid in each well is the dominant factor in determining theheat capacity of each sample and, therefore, is the dominant factor inthe thermal time constant for that particular sample well.

Note that the ability to cause the sample liquid in all the sample wellsto cycle up and down in temperature in unison and to stabilize at targettemperatures very near each other, i.e., in tolerance bands that areonly 0.5° C. wide, also depends upon the force F in FIG. 15. This forcemust exceed a minimum threshold force before the thermal time constantsof all sample wells loaded with similar masses of sample liquid willhave the same time constant. This minimum threshold force has beenexperimentally determined to be 30 grams for the sample tube and samplewell configuration described herein. For higher levels of accuracy, theminimum threshold force F in FIG. 15 should be established at at least50 grams and preferably 100 grams for an additional margin of safety asnoted above.

The importance of thermal uniformity in sample well temperature can beappreciated by reference to FIG. 18. This figure shows the relationshipbetween the amount of DNA generated in a PCR cycle and the actual sampletemperature during the denaturation interval for one instance ofamplification of a certain segment of DNA. The slope of function 298between temperatures 93 and 95 degrees centigrade is approximately 8%per degree centigrade for this particular segment of DNA and primers.FIG. 18 shows the general shape of the curve which relates the amount ofDNA generated by amplification, but the details of the shape of thecurve vary with every different case of primers and DNA target.Temperatures for denaturation above 97 degrees centigrade are generallytoo hot and result in decreasing amplification for increasingdenaturation temperature. Temperatures between 95 and 97 degreescentigrade are generally just right.

FIG. 18 illustrates that any sample well containing this particular DNAtarget and primer combination which stabilizes at a denaturationtemperature of approximately 93° C. is likely to have 8% less DNAgenerated over the course of a typical PCR protocol than wells denaturedat 94° C. Likewise, sample liquids of this mixture that stabilize atdenaturation temperatures of 95° C. are likely to have 8% more DNAgenerated therein than is generated in sample wells which stabilize atdenaturation temperatures of 94° C. Because all curves of this naturehave the same general shape, it is important to have uniformity insample temperature.

The sample temperatures calculated as described above are used by thecontrol algorithm for controlling the heaters and flow through the rampcooling channels and to determine how long the samples have been held atvarious target temperatures. The control algorithm uses these times forcomparison with the desired times for each incubation period as enteredby the user. When the times match, the control algorithm takes theappropriate steps to heat or cool the sample block toward the targettemperature defined by the user for the next incubation.

When the calculated sample temperature is within one degree centigradeof the setpoint, i.e., the incubation temperature programmed by theuser, the control program causes a timer to start. This timer may bepreset to count down from a number set so as to time out the intervalspecified by the user for the incubation being performed. The timerstarts to count down from the preset count when the calculated sampletemperature is within one degree centigrade. When the timer reaches azero count, a signal is activated which causes the CPU to take actionsto implement the next segment of the PCR protocol. Any way to time thespecified interval will suffice for purposes of practicing theinvention.

Typically, the tolerance band around any particular target temperatureis plus or minus 0.5° C. Once the target temperature is reached, thecomputer holds the sample block at the target temperature using the biascooling channels and the film heater such that all the samples remainclose to the target temperature for the specified interval.

For the thermal system described herein to work well, the thermalconductance from the sample block to each sample must be known anduniform to within a very close tolerance. Otherwise, not all sampleswill be held within the specified tolerance band of the targettemperature when the timer starts and, not all the samples willexperience the same incubation intervals at the target temperature.

Also, for this thermal system to work well, all sample tubes must beisolated from variables in the ambient environment. That is, it isundesirable for some sample tubes to be cooled by drafts while othersample tubes in different physical positions do not experience the samecooling effects. For good uniformity it is highly desirable that thetemperatures of all the samples be determined by the temperature of thesample block and by nothing else.

Isolation of the tubes from the ambient, and application of the minimumthreshold force F pushing down on the sample tubes is achieved by aheated cover over the sample tubes and sample block.

Even though the sample liquid is in a sample tube pressed tightly into atemperature-controlled metal block, tightly capped, with a meniscus wellbelow the surface of the temperature-controlled metal block, the samplesstill lose their heat upward by convection. Significantly, when thesample is very hot (the denaturation temperature is typically near theboiling point of the sample liquid), the sample liquid can lose a verysignificant amount of heat by refluxing of water vapor. In this process,water evaporates from the surface of the hot sample liquid and condenseson the inner walls of the cap and the cooler upper parts of the sampletube above the top surface of the sample block. If there is a relativelylarge volume of sample, condensation continues, and condensate builds upand runs back down the walls of the sample tube into the reactionmixture. This "refluxing" process carries about 2300 joules of heat pergram of water refluxed. This process can cause a drop of several degreesin the surface temperature of a 100 microliter reaction mixture therebycausing a large reduction of efficiency of the reaction.

If the reaction mixture is small, say 20 microliters, and the sampletube has a relatively large surface area above the top surface of thesample block, a significant fraction of the water in the reactionmixture may evaporate. This water may then condense inside the upperpart of the sample tube and remain there by surface tension during theremainder of the high temperature part of the cycle. This can soconcentrate the remaining reaction mixture that the reaction is impairedor fails completely.

In the prior art PCR thermal cyclers, this refluxing problem was dealtwith by overlaying the reaction mixture with a layer of oil or meltedwax. This immiscible layer of oil or wax floated on the aqueous reactionmixture and prevented rapid evaporation. However, labor was required toadd the oil which raised processing costs. Further, the presence of oilinterfered with later steps of processing and analysis and created apossibility of contamination of the sample. In fact, it is known thatindustrial grade mineral oils have in the past contaminated samples bythe unknown presence of contaminating factors in the oil which wereunknown to the users.

The need for an oil overlay is eliminated, and the problems of heat lossand concentration of the reaction mixture by evaporation andunpredictable thermal effects caused by refluxing are avoided accordingto the teachings of the invention by enclosing the volume above thesample block into which the upper parts of the sample tubes project andby heating this volume from above by a heated cover sometimes hereafteralso called the platen.

Referring to FIG. 19, there is shown a cross sectional view of thestructure which is used to enclose the sample tubes and apply downwardforce thereto so as to supply the minimum threshold force F in FIG. 15.A heated platen 14 is coupled to a lead screw 312 so as to move up anddown along the axis symbolized by arrow 314 with rotation of the leadscrew 312. The lead screw is threaded through an opening in a slidingcover 316 and is turned by a knob 318. The platen 314 is heated to atemperature above the boiling point of water by resistance heaters (notshown) controlled by computer 20.

The sliding cover 316 slides back and forth along the Y axis on rails320 and 322. The cover 316 includes vertical sides 317 and 319 and alsoincludes vertical sides parallel to the X-Z plane (not shown) whichenclose the sample block 12 and sample tubes. This structuresubstantially prevent drafts from acting on the sample tubes of whichtubes 324 and 326 are typical.

FIG. 20 is a perspective view of the sliding cover 316 and sample block12 with the sliding cover in retracted position to allow access to thesample block. The sliding cover 316 resembles the lid of a rectangularbox with vertical wall 328 having a portion 330 removed to allow thesliding cover 316 to slide over the sample block 12. The sliding coveris moved along the Y axis in FIG. 20 until the cover is centered overthe sample block 12. The user then turns the knob 318 in a direction tolower the heated platen 14 until a mark 332 on the knob 318 lines upwith a mark 334 on an escutcheon plate 336. In some embodiments, theescutcheon plate 336 may be permanently affixed to the top surface ofthe sliding cover 316. In other embodiments, the escutcheon 336 may berotatable such that the index mark 334 may be placed in differentpositions when different size sample tubes are used. In other words, iftaller sample tubes are used, the heated platen 14 need not be loweredas much to apply the minimum threshold force F in FIG. 15. In use, theuser screws the screw 318 to lower the platen 14 until the index marksline up. The user then knows that the minimum threshold force F willhave been applied to each sample tube.

Referring jointly to FIGS. 15 and 19, prior to lowering the heatedplaten 14 in FIG. 19, the plastic cap 338 for each sample tube sticks upabout 0.5 millimeters above the level of the top of the walls of aplastic tray 340 (FIG. 19) which holds all the sample tubes in a loose8×12 array on 9 millimeter centers. The array of sample wells can holdup to 96 MicroAmp™ PCR tubes of 100 μL capacity or 48 larger GeneAmp™tubes of 0.5 ml capacity. The details of this tray will be discussed ingreater detail below. The tray 340 has a planar surface having an 8×12array of holes for sample tubes. This planar surface is shown in FIGS.15 and 19 as a horizontal line which intersects the sample tubes 324 and326 in FIG. 19. Tray 340 also has four vertical walls two of which areshown at 342 and 344 in FIG. 19. The top level of these vertical walls,shown at 346 in FIG. 15, establishes a rectangular box which defines areference plane.

As best seen in FIG. 15, the caps 338 for all the sample tubes projectabove this reference plane 346 by some small amount which is designed toallow the caps 338 to be softened and deformed by the heated platen 14and "squashed" down to the level of the reference plane 346. In thepreferred embodiment, the heated platen 14 is kept at a temperature of105° C. by the CPU 20 in FIG. 1 and the bus 22 coupled to resistanceheaters (not shown) in the platen 14. In the preferred embodiment, theknob 318 in FIG. 19 and the lead screw 312 are turned until the heatedplaten 14 descends to and makes contact with the tops of the caps 338.In the preferred embodiment, the caps 338 for the sample tubes are madeof polypropylene. These caps soften shortly after they come into contactwith the heated platen 14. As the caps soften, they deform, but they donot lose all of their elasticity. After contacting the caps, the heatedplaten is lowered further until it rests upon the reference plane 346.This further lowering deforms the caps 338 and causes a minimumthreshold force F of at least 50 grams to push down on each sample tubeto keep each tube well seated firmly in its sample well. The amount bywhich the caps 338 project above the reference plane 346, and the amountof deformation and residual elasticity when the heated platen 14 restsupon the reference plane 346 is designed such that a minimum thresholdforce F of at least 50 grams and preferably 100 grams will have beenachieved for all sample tubes then present after the heated platen 14has descended to the level of the reference plane 346.

The heated platen 14 and the four vertical walls and planar surface ofthe tray 340 form a heated, sealed compartment when the platen 14 is incontact with the top edge 346 of the tray. The plastic of the tray 340has a relatively poor thermal conductivity property. It has been foundexperimentally that contacting the heated platen 14 with the caps 338and the isolation of the portion of the sample tubes 288 which projectabove the top level 280 of the sample block 12 by a wall of materialwhich has relatively poor thermal conductivity has a beneficial result.With this structure, the entire upper part of the tube and cap arebrought to a temperature which is high enough that little or nocondensation forms on the inside surfaces of the tube and cap since theheated platen is kept at a temperature above the boiling point of water.This is true even when the sample liquid 276 in FIG. 15 is heated to atemperature near its boiling point. This eliminates the need for a layerof immiscible material such as oil or wax floating on top of the samplemixture 276 thereby reducing the amount of labor involved in a PCRreaction and eliminating one source of possible contamination of thesample.

It has been found experimentally that in spite of the very hightemperature of the heated cover and its close proximity to the sampleblock 12, there is little affect on the ability of the sample block 12to cycle accurately and rapidly between high and low temperatures.

The heated platen 14 prevents cooling of the samples by the refluxingprocess noted earlier because it keeps the temperature of the caps abovethe condensation point of water thereby keeping the insides of the capsdry. This also prevents the formation of aerosols when the caps areremoved from the tubes.

In alternative embodiments, any means by which the minimum acceptabledownward force F in FIG. 15 can be applied to each individual sampletube regardless of the number of sample tubes present and which willprevent condensation and refluxing and convection cooling will sufficefor purposes of practicing the invention. The application of thisdownward force F and the use of heat to prevent refluxing and undesiredsample liquid concentration need not be both implemented by the samesystem as is done in the preferred embodiment.

The sample tubes may vary by a few thousandths of an inch in theiroverall height. Further, the caps for the sample tubes may also vary inheight by a few thousandths of an inch. Also, each conical sample wellin the sample block 12 may not be drilled to exactly the same depth, andeach conical sample well in the sample block may be drilled to aslightly different diameter and angle. Thus, when a population of cappedtubes is placed in the sample block so as to be seated in thecorresponding sample well, the tops of the caps will not all necessarilybe at the same height. The worst case discrepancy for this height couldbe as much as 0.5 millimeters from the highest to the lowest tubes.

If a perfectly flat unheated platen 14 mounted so that it is free tofind its own position were to be pressed down on such an array of caps,it would first touch the three tallest tubes. As further pressure wasapplied and the tallest tubes were compressed somewhat, the platen wouldbegin to touch some caps of lower tubes. There is a distinct possibilitythat unless the tube and cap assemblies were compliant, the tallesttubes would be damaged before the shortest tubes were contacted at all.Alternatively, the force necessary to compress all the tall tubessufficiently so as to contact the shortest tube could be too large forthe device to apply. In either case, one or more short tubes might notbe pressed down at all or might be pressed down with an insufficientamount of force to guarantee that the thermal time constant for thattube was equal to the thermal time constants for all the other tubes.This would result in the failure to achieve the same PCR cycle for alltubes in the sample block since some tubes with different thermal timeconstants would not be in step with the other tubes. Heating the platenand softening the caps eliminates these risks by eliminating themanufacturing tolerance errors which lead to differing tube heights as afactor.

In an alternative embodiment, the entire heated platen 14 is coveredwith a compliant rubber layer. A compliant rubber layer on the heatedplaten would solve the height tolerance problem, but would also act as athermal insulation layer which would delay the flow of heat from theheated platen to the tube caps. Further, with long use at hightemperatures, most rubber materials deteriorate or become hard. It istherefore desirable that the heated platen surface be a metal and a goodconductor of heat.

In another alternative embodiment, 96 individual springs could bemounted on the platen so that each spring individually presses down on asingle sample tube. This is a complex and costly solution, however, andit requires that the platen be aligned over the tube array with amechanical precision which would be difficult or bothersome to achieve.

The necessary individual compliance for each sample tube in thepreferred embodiment is supplied by the use of plastic caps whichcollapse in a predictable way under the force from the platen but which,even when collapsed, still exert a downward force F on the sample tubeswhich is adequate to keep each sample tube seated firmly in its well.

In the sample tube cap 338 shown in FIG. 15, the surface 350 should befree of nicks, flash and cuts so that it can provide a hermetic sealwith the inner walls 352 of the sample tube 288. In the preferredembodiment, the material for the cap is polypropylene. A suitablematerial might be Valtec HH-444 or PD701 polypropylene manufactured byHimont as described above or PPW 1780 by American Hoescht. As shown inFIG. 15, in the preferred embodiment, the wall thickness for the domedportion of the cap is about the same as the wall thickness of theadjacent tube portion, most preferably 0.018-0.022 inches. The thicknessof the shoulder portion 356 is 0.025 inches and the width of the domedshaped portion of the cap is 0.203 inches in the preferred embodiment.

Any material and configuration for the caps which will cause the minimumthreshold force F in FIG. 15 to be applied to all the sample tubes andwhich will allow the cap and upper portions of the sample tubes to beheated to a temperature high enough to prevent condensation andrefluxing will suffice for purposes of practicing the invention. Thedome shaped cap 338 has a thin wall to aid in deformation of the cap.Because the heated platen is kept at a high temperature, the wallthickness of the domed shaped cap can be thick enough to be easilymanufactured by injection molding since the necessary compliance toaccount for differences in tube height is not necessary at roomtemperature.

The platen can be kept at a temperature anywhere from 94° C. to 110° C.according to the teachings of the invention although the range from 100°C. to 110° C. is preferred to prevent refluxing since the boiling pointof water is 100° C. In this temperature range, it has beenexperimentally found that the caps soften just enough to collapse easilyby as much as 1 millimeter. Studies have shown that the elasticproperties of the polypropylene used are such that even at thesetemperatures, the collapse is not entirely inelastic. That is, eventhough the heated platen causes permanent deformation of the caps, thematerial of the caps still retain a significant enough fraction of theirroom temperature elastic modulus that the minimum threshold force F isapplied to each sample tube. Further, the heated platen levels all thecaps that it contacts without excessive force regardless of how manytubes are present in the sample block because of the softening of thecap.

Because the cap temperature is above the boiling point of water duringthe entire PCR cycle, the inside surfaces of each cap remain completelydry. Thus, at the end of a PCR process, if the samples are cooled toroom temperature before being removed from the sample block, if the capson each sample tube are opened, there is no possibility of creating anaerosol spray of the sample tube contents which could result in crosscontamination. This is because there is no liquid at the cap to tubeseal when the seal is broken.

This is extremely advantageous, because tiny particles of aerosolcontaining amplified product DNA can contaminate a laboratory and getinto sample tubes containing samples from other sources, e.g., otherpatients, thereby possibly causing false positive or negative diagnosticresults which can be very troublesome. Users of the PCR amplificationprocess are extremely concerned that no aerosols that can contaminateother samples be created.

A system of disposable plastic items is used to convert the individualsample tubes to an 8×12 array which is compatible with microtiter plateformat lab equipment but which maintains sufficient individual freedomof movement to compensate for differences in the various rates ofthermal expansion of the system components. The relationship of thethermally compliant cap to the rest of this system is best seen in FIG.21A which is a cross sectional view of the sample block, and two sampletubes with caps in place with the sample tubes being held in place bythe combination of one embodiment of a plastic 96 well microtiter trayand a retainer. FIG. 21B is an alternative, preferred embodiment showingthe structure and interaction of most of the various plastic disposableitems of the system. The rectangular plastic 96 well microtiter platetray 342 rests on the surface of the sample block 12. The top edge 346of the frame 342 has a height which is approximately 0.5 millimetersshorter than the height of the caps of which cap 364 is exemplary. Allof the capped tubes will project higher than the edge 346 of the frame342. The frame 342 is configured such that a downward extending ridge366 extends into the guardband groove 78 through its entire length. Theframe 342 does however have a gap (not shown) which corresponds to thegap in the groove 78 for the temperature sensor shown in FIG. 2 in planview and in FIG. 7 in cross-sectional view.

The reference plane 346 mentioned above is established by the top of theframe 342. How this reference plane interacts with the heated platen isas follows. Prior to screwing down the knob 318 in FIG. 20 to line upthe index marks 332 and 334 to start an amplification run, a calibrationprocess will have been performed to locate the position of the indexmark on the escutcheon platen 336 in FIG. 20. This calibration isstarted by placing the frame 342 in FIG. 21 in position on the sampleblock. The frame 342 will be empty however or any sample tubes thereinwill not have any caps in place. Then, the knob 318 is screwed downuntil the heated platen 14 is firmly in contact with the top edge 346 ofthe frame 342 around its entire parameter. When the knob 318 has beenscrewed down sufficiently to allow the heated platen to rest on thereference plane 346 and to press the frame 342 firmly against the topsurface 280 of the sample block, the rotatable escutcheon 336 of thepreferred embodiment will be rotated until the index mark 334 on theescutcheon lines up with the index mark 332 on the knob 318. Then, theknob 318 is rotated counterclockwise to raise the platen 14 and thecover 316 in FIG. 19 is slid in the negative Y direction to uncover theframe 342 and the sample block 12. Sample tubes with caps loaded with asample mixture may then be placed in position in the frame 342. Theheated cover 316 is then placed back over the sample block, and the knob318 is turned clockwise to lower the heated platen 14 until the indexmark 332 on the knob lines up with the index mark 334 as previouslypositioned. This guarantees that all tubes have been firmly seated withthe minimum force F applied. The use of the index marks gives the user asimple, verifiable task to perform.

If there are only a few sample tubes in place, it will take only a smallamount of torque to line up the index marks 332 and 334. If there aremany tubes, however, it will take more torque on the knob 318 to line upthe index marks. This is because each tube is resisting the downwardmovement of the heated platen 14 as the caps deform. However, the useris assured that when the index marks 332 and 334 are aligned, the heatedplaten will once again be tightly placed against the top edge 346 of theframe 342 and all tubes will have the minimum threshold force F appliedthereto. This virtually guarantees that the thermal time constant forall the tubes will be substantially the same.

In alternative embodiments, the index marks 332 and 334 may be dispensedwith, and the knob 318 may simply be turned clockwise until it will notturn any more. This condition will occur when the heated platen 314 hasreached the top edge or reference plane 346 and the plastic frame 342has stopped further downward movement of the heated platen 14. Obviouslyin this alternative embodiment, and preferably in the index markembodiment described above, the plastic of the frame 342 will have amelting temperature which is sufficiently high to prevent deformation ofthe plastic of the frame 342 when it is in contact with the heatedplaten 14. In the preferred embodiment, the plastic of the frame 342 iscelanese nylon 1503 with a wall thickness of 0.05 inches.

An advantage of the above described system is that sample tubes ofdifferent heights may be used simply by using frames 342 havingdifferent heights. The frame 342 should have a height which isapproximately 0.5 millimeters shorter than the plane of the tips of thecapped tubes when both are seated in the sample block. In the preferredembodiment, two different tube heights are used. The range of motion ofthe lead screw 312 which drives the heated platen 14 in FIG. 19 must besufficient for all the different sizes of sample tubes to be used. Ofcourse, during any particular PCR processing cycle, all tubes must bethe same height.

The system described above provides uniform temperatures in the sampleblock, uniform thermal conductance from block to sample, and isolationof the sample tubes from the vagaries of the ambient environment. Anynumber of sample tubes up to 96 may be arrayed in the microtiter plateformat. The system allows accurate temperature control for a very largenumber of samples and a visual indication of the sample temperatures forall samples without actually measuring the temperature of any sample.

As the container for PCR reactions, it has been common in the prior artto use polypropylene tubes which were originally designed formicrocentrifuges. This prior art tube had a cylindrical cross-sectionclosed at the top by a snap-on cap which makes a gas-tight seal. Thisprior art tube had a bottom section which comprised the frustum of acone with an included angle of approximately 17 degrees.

When such a conical sample tube is pressed down into a sample well of asample block with a conical cavity with the same included angle, andwhen the sample mixture in the tube lies entirely within the conicalvolume and below the top surface of the sample block, the thermalconductance between the block and the liquid can be made adequatelypredictable for good uniformity of sample temperature throughout thearray. To achieve adequate control of the thermal conductance betweenthe sample block and the sample mixture, the included angles of theconical tube and the sample well must match closely, and the conicalsurfaces of the tube and well must be smooth and held together in flushrelation. Further, the minimum threshold force F must be applied to eachsample tube to press each tube tightly into the sample well so that itdoes not rise up or loosen in the well for any reason during thermalcycling, such as steam formation from trapped liquid in space 291 inFIG. 15. Finally, each tube must be loaded with the same amount ofsample liquid. If the above listed conditions are met, the thermalconductance between the sample block and the sample liquid in each tubewill be predominantly determined by the conductance of the conicalplastic wall 368 in FIG. 15 and a boundary layer, (not shown) of thesample liquid at the inside surface 370 of the conical sample tube wall.

The thermal conductance of the plastic tube walls is determined by theirthickness, which can be closely controlled by the injection moldingmethod of manufacture of the tubes. The sample liquid in all the sampletubes has virtually identical thermal properties.

It has been found by experiment and by calculation that a molded,one-piece, 96-well microtiter plate is only marginally feasible for PCRbecause the differences in the thermal expansion coefficients betweenaluminum and plastic lead to dimensional changes which can destroy theuniformity of thermal conductance to the sample liquid across the array.That is, since each well in such a one-piece plate is connected to eachother well through the surface of the plate, the distances between thewells are determined at the time of initial manufacture of the plate butchange with changing temperature since the plastic of the plate has asignificant coefficient of thermal expansion. Also, distances betweenthe sample wells in the metal sample block 12 are dependent upon thetemperature of the sample block since aluminum also has a significantcoefficient of thermal expansion which is different than that ofplastic. To have good thermal conductance, each sample well in aone-piece 96-well microtiter plate would have to fit almost perfectly inthe corresponding well in the sample block at all temperatures. Sincethe temperature of the sample block changes over a very wide range oftemperatures, the distances between the sample wells in the sample blockvary cyclically during the PCR cycle. Because the coefficients ofthermal expansion for plastic and aluminum are substantially different,the distances of the well separation in the sample block would varydifferently over changing temperatures than would the distances betweenthe sample wells of a plastic, one-piece, 96-well microtiter plate.

Thus, as an important criteria for a perfect fit between a sample tubeand the corresponding sample well over the PCR temperature range, it isnecessary that each sample tube in the 96-well array be individuallyfree to move laterally and each tube must be individually free to bepressed down vertically by whatever amount is necessary to make flushcontact with the walls of the sample well.

The sample tubes used in the invention are different from the prior artmicrocentrifuge tubes in that the wall thickness of the conical frustumposition of the sample tube is much thinner to allow faster heattransfer to and from the sample liquid. The upper part of these tubeshas a thicker wall thickness than the conical part. In FIG. 15, the wallthickness in the cylindrical part 288 in FIG. 15 is generally 0.030inches while the wall thickness for the conical wall 368 is 0.009inches. In a preferred embodiment, the wall thickness in the cylindricalpart above shoulder 384 is about 0.022 inches, the wall thickness in thecylindrical part below shoulder 384 is about 0.015 inches, and the wallthickness in the conical section is in the range 0.009±0.001 inchesaverage, although intra-wall variations can vary from nominal by up tofour times that amount. Because thin parts cool faster than thick partsin the injection molding process, it is important to get the mold fullbefore the thin parts cool off.

The material of the sample tubes must be compatible chemically with thePCR reaction. Glass is not a PCR compatible material, because DNA sticksto glass and will not come off which would interfere with PCRamplification. Preferably an autoclavable polypropylene is used. Threetypes of suitable polypropylene were identified earlier herein. Someplastics are not compatible with the PCR process because of outgassingof materials from the plastic or because DNA sticks to the plasticwalls. Polypropylene is the best known class of plastics at this time.

Conventional injection molding techniques and mold manufacturetechniques for the injection mold will suffice for purposes ofpracticing the invention.

The use of cone shaped sample tubes translates substantially allmanufacturing tolerance errors to height errors, i.e., a variance fromtube to tube in the height of the tip of the cap to the top of thesample block when the sample tube is seated in the sample well. Forexample, an angle error for the angle of the sample tube walls isconverted to a height error when the tube is placed in the sample blockbecause of the mismatch between the tube wall angle and the sample wellwall angle. Likewise, a diameter error in the dimensions of the conewould also translate into a height error since the conical part of thetube would either penetrate deeper or not as much as a properlydimensional tube.

For good uniformity of thermal conductance across the array, a good fitbetween the sample tubes and the sample well must exist for all 96-wellsover the full temperature range of 0° to 100° C. regardless ofdifferences in thermal expansion rates. Also, each of the 96 sampletubes must have walls with dimensions and wall thicknesses which areuniform to a very high degree. Each sample tube in which sample mixtureis to be held should be fitted with a removable gas-tight cap that makesa gas-tight seal to prevent loss of water vapor from the reactionmixture when this mixture is at or near its boiling point such that thevolume of the sample mixture does not decrease. All these factorscombine to make a one-piece microtiter plate with 96 individual samplewells extremely difficult to manufacture in a manner so as to achieveuniform thermal conductance for all 96 wells.

Any structure which provides the necessary individual lateral andvertical degrees of freedom for each sample tube will suffice forpurposes of practicing the invention.

According to the teachings of the preferred embodiment of the invention,all the above noted requirements have been met by using a 4 piecedisposable plastic system. This system gives each sample tube sufficientfreedom of motion in all necessary directions to compensate fordiffering rates of thermal expansion and yet retains up to 96 sampletubes in a 96 well microtiter plate format for user convenience andcompatibility with other laboratory equipment which is sized to workwith the industry standard 96-well microtiter plate. The multi-piecedisposable plastic system is very tolerant of manufacturing toleranceerrors and the differing thermal expansion rates over the widetemperature range encountered during PCR thermal cycling.

FIGS. 21A and 21B show alternative embodiments of most of the four pieceplastic system components in cross-section as assembled to hold aplurality of sample tubes in their sample wells with sufficient freedomof motion to account for differing rates of thermal expansion. FIG. 45shows all the parts of the disposable plastic microtiter plate emulationsystem in an exploded view. This figure illustrates how the parts fittogether to form a microtiter plate with all the sample tubes looselyretained in an 8×12 microtiter plate format 96 well array. FIG. 22 showsa plan view of a microtiter plate frame 342 according to the teachingsof the invention which is partially shown in cross-section in FIGS. 21Aand 21B. FIG. 23 shows a bottom view plan view of the frame 342. FIG. 24is an end view of the frame 342 taken from view line 24-24' in FIG. 22.FIG. 25 is an end view of the frame 342 taken from view line 25-25' inFIG. 22. FIG. 26 is a cross section through the frame 342 at sectionline 26-26' in FIG. 22. FIG. 27 is a cross sectional view through theframe 342 taken along section line 27-27' in FIG. 22. FIG. 28 is a sideview of the frame 342 taken along view line 28-28' in FIG. 22 with apartial cut away to show in more detail the location where a retainer tobe described below clips to the frame 342.

Referring jointly to FIGS. 21A, 21B and 22 through 28, the frame 342 iscomprised of a horizontal plastic plate 372 in which there are formed 96holes spaced on 9 millimeter centers in the standard microtiter plateformat. There are 8 rows labeled A through H and 12 columns labeled 1through 12. Hole 374 at row D, column 7 is typical of these holes. Ineach hole in the frame 342 there is placed a conical sample tube such asthe sample tube 376 shown in FIG. 15. Each sample tube is smaller indiameter than the hole in which it is placed by about 0.7 millimeters,so that there is a loose fit in the hole. This is best seen in FIGS. 21Aand 21B by observing the distance between the inside edge 378 of atypical hole and the side wall 380 of the sample tube placed therein.Reference numeral 382 in FIGS. 21A and 21B shows the opposite edge ofthe hole which is also spaced away from the outside wall of thecylindrical portion of the sample tube 376.

Each sample tube has a shoulder shown at 384 in FIGS. 15, 21A and 21B.This shoulder is molded around the entire circumference of thecylindrical portion 288 of each sample tube. As is shown in a preferredembodiment of FIGS. 21A, 21B and 26, the lower surface of shoulder 384is beveled and the hole 374 is countersunk. This aids in centering thetube upright in hole 374 in frame 342. The diameter of this shoulder 384is large enough that it will not pass through the holes in the frame342, yet not so large as to touch the shoulders of the adjacent tubes inneighboring holes.

Once all the tubes are placed in their holes in the frame 42, a plasticretainer 386 (best seen in FIGS. 21A and 21B FIG. 45) is snapped intoapertures in the frame 342. The purpose of this retainer is to keep allthe tubes in place such that they cannot fall out or be knocked out ofthe frame 342 while not interfering with their looseness of fit in theframe 342. The retainer 386 is sized and fitted to the frame 342 suchthat each sample tube has freedom to move vertically up and down to someextent before the shoulder 384 of the tube encounters either theretainer 386 or the frame 342. Thus, the frame and retainer, whencoupled, provide a microtiter plate format for up to 96 sample tubes butprovide sufficient horizontal and vertical freedom such that each tubeis free to find its best fit at all temperatures under the influence ofthe minimum threshold force F in FIG. 15. As shown in the embodimentdepicted in FIG. 15, shoulder 384 may be located approximately in themidsection of cylindrical portion 288 of tube 376.

A more clear view of the sample tube and shoulder may be had byreference to FIGS. 29 and 30. FIGS. 29 and 30 are an elevation sectionalview and a partial upper section of the shoulder portion, respectively,of a typical sample tube. A plastic dome-shaped cap such as will bedescribed in more detail below is inserted into the sample tube shown inFIG. 29 and forms a hermetic seal with the inside wall 390 of the top atthe sample tube. A ridge 392 formed in the inside wall of the sampletube acts as a stop for the dome-shaped cap to prevent furtherpenetration. Normally, the dome-shaped caps come in strips connected byweb.

FIG. 31 shows three caps in elevation view connected by a web 394 andterminated in a tab 396. The tab aids the user in removing an entire rowof caps by a single pull. Normally, the web 394 rests on the top surface398 of the sample tube and prevents further penetration of the cap intothe sample tube. Each cap includes a ridge 400 which forms the hermeticseal between the cap and the inside wall of the sample tube. As shownparticularly in FIGS. 30 and 31, the bottom outside of the cap side orridge 400 may be beveled, the top of inner wall 390 of the tube may beflared, or both. This aids in inserting the cap into the tube. FIG. 32shows a top view of three caps in a typical strip of 12 connected caps.

For a more detailed understanding of the retainer, refer to FIGS. 33through 37. FIG. 33 is a top view of the plastic retainer. FIG. 34 is anelevation view of the retainer taken along view line 34-34' in FIG. 33.FIG. 35 is an end elevation view of the retainer taken along view line35-35' in FIG. 33. FIG. 36 is a sectional view taken along section line36-36''in FIG. 33. FIG. 37 is a sectional view through the retainertaken along section line 37-37' in FIG. 33.

Referring jointly to FIGS. 33-37, the retainer 386 is comprised of asingle horizontal plastic plane 402 surrounded by a vertical wall 404.The plane 402 has an 8×12 array of 96 holes formed therein divided into24 groups of four holes per group. These groups are set off by ridgesformed in the plane 402 such as ridges 406 and 408. Each hole, of whichhole 410 is typical, has a diameter D which is larger than the diameterD₁ in FIG. 29 and smaller than the diameter D₂. This allows the retainerto be slipped over the sample tubes after they have been placed in theframe 342 but prevents the sample tubes from falling out of the framesince the shoulder 384 is too large to pass through the hole 410.

The retainer snaps into the frame 342 by means of plastic tabs 414 shownin FIGS. 34 and 36. These plastic tabs are pushed through the slots 416and 418 in the frame as shown in FIG. 23. There are two plastic tabs414, one on each long edge of the retainer. These two plastic tabs areshown as 414A and 414B in FIG. 33.

The frame 342 of FIGS. 22-28, with up to 96 sample tubes placed thereinand with the retainer 386 snapped into place, forms a single unit suchas is shown in FIGS. 21A and 21B which can be placed in the sample block12 for PCR processing.

After processing, all the tubes may be removed simultaneously by liftingthe frame 342 out of the sample block. For convenience and storage, theframe 342 with sample tubes and retainer in place can be inserted intoanother plastic component called the base. The base has the outsidedimensions and footprint of a standard 96-well microtiter plate and isshown in FIGS. 38 through 44. FIG. 38 is a top plan view of the base420, while FIG. 39 is a bottom plan view of the base. FIG. 40 is anelevation view of the base taken from view line 40-40' in FIG. 38. FIG.41 is an end elevation view taken from view line 41-41' in FIG. 38. FIG.42 is a sectional view taken through the base along section line 42-42'in FIG. 38. FIG. 43 is a sectional view through the base taken alongsection line 43-43' in FIG. 38. FIG. 44 is a sectional view taken alongsection line 44-44' in FIG. 38.

The base 420 includes a flat plane 422 of plastic in which an 8×12 arrayof holes with sloped edges is formed. These holes have dimensions andspacing such that when the frame 342 is seated in the base, the bottomsof the sample tubes fit into the conical holes in the base such that thesample tubes are held in the same relationship to the frame 342 as thesample tubes are held when the frame 342 is mounted on the sample block.Hole 424 is typical of the 96 holes formed in the base and is shown inFIGS. 38, 44 and 43. The individual sample tubes, though looselycaptured between the tray and retainer, become firmly seated andimmobile when the frame is inserted in the base. The manner in which atypical sample tube 424 fits in the base is shown in FIG. 44.

In other words, when the frame, sample tubes and retainer are seated inthe base 420 the entire assembly becomes the exact functional equivalentof an industry standard 96-well microtiter plate, and can be placed invirtually any automated pipetting or sampling system for 96-wellindustry standard microtiter plates for further processing.

After the sample tubes have been filled with the necessary reagents andDNA sample to be amplified, the sample tubes can be capped. In analternative embodiment of the cap strip shown in FIGS. 31 and 32, anentire mat of 96 caps with a compliant web connecting them in an 8×12array may be used. This web, shown at 394 in FIG. 31 must besufficiently compliant so that the caps do not restrain the sample tubesfrom making the small motions these sample tubes must make to fitperfectly in the conical wells of the sample block at all temperatures.

The assembly of tubes, caps, frames, retainer and base is brought afterfilling the tubes to the thermal cycler. There, the frame, capped tubesand retainer plate are removed from the base as a unit. This unit isthen placed in the sample block 12 to make the assembly shown in FIG.21A or 21B with the tubes loosely held in the conical wells in thesample block. As shown in FIG. 21, the frame 342 is seated on the topsurface 280 of the guardband. In the preferred embodiment, the ridge 366extends down into the groove 78 of the guardband, but this is notessential.

Next, the heated cover is slid over the samples, and the heated platenis screwed down as previously described until it contacts the top edge346 of the frame 342.

Within seconds after the heated platen 14 in FIG. 19 touches the caps,the caps begin to soften and yield under the downward pressure from thelead screw 312 in FIG. 19. The user then continues to turn to knob 318until the index marks 332 and 334 in FIG. 20 line up which indicatesthat every sample tube has been tightly pressed into the sample blockwith at least the minimum threshold force F and all air gaps between theheated platen 14, the sample block and the top edge 346 of the frame 342have been tightly closed. The sample tubes are now in a completelyclosed and controlled environment, and precision cycling of temperaturecan begin.

At the end of the PCR protocol, the heated platen 14 is moved upward andaway from the sample tubes, and the heated cover 316 is slid out of theway to expose the frame 342 and sample tubes. The frame, sample tubesand retainer are then removed and replaced into an empty base, and thecaps can be removed. As each cap or string of caps is pulled off, theretainer keeps the tube from coming out of the tray. Ribs formed in thebase (not shown in FIGS. 38-44) contact the retainer tabs 414A and 414Bshown in FIG. 33 to keep the retainer snapped in place such that theforce exerted on the tubes by removing the caps does not dislodge theretainer 386.

Obviously, the frame 342 may be used with fewer than 96 tubes ifdesired. Also, the retainer 386 can be removed if desired by unsnappingit.

A user who wishes to run only a few tubes at a time and handle thesetubes individually can place an empty frame 342 without retainer on thesample block. The user may then use the base as a "test tube rack" andset up a small number of tubes therein. These tubes can then be filledmanually and capped with individual caps. The user may then transfer thetubes individually into wells in the sample block, close the heatedcover and screw down the heated platen 14 until the marks line up. PCRcycling may then commence. When the cycling is complete, the cover 316is removed and the sample tubes are individually placed in an availablebase. The retainer is not necessary in this type of usage.

Referring to FIG. 50, there is shown a cross-sectional view of a largervolume, thin walled reaction tube marketed under the trademark MAXIAMP.This tube is useful for PCR reactions wherein reagents or othermaterials need to be added to the reaction mixture which will bring thetotal volume to greater than 200 microliters. The larger tube shown inFIG. 50 made of Himont PD701 polypropylene or Valtec HH-444polypropylene and has a thin wall in contact with the sample block.Whatever material is selected should be compatible with the DNA andother components of the PCR reaction mixture so as to not impair PCRreaction processing such as by having the target DNA stick to the wallsand not replicate. Glass is generally not a good choice because DNA hasbeen known to stick to the walls of glass tubes.

The dimension A in FIG. 50 is typically 0.012±0.001 inches (half thethickness of cylindrical wall section D) and the wall angle relative tothe longitudinal axis of the tube is typically 17°. The advantage of a17° wall angle is that while downward force causes good thermal contactwith the sample block, the tubes do not jam in the sample wells. Theadvantage of the thin walls is that it minimizes the delay betweenchanges in temperature of the sample block and corresponding changes intemperature of the reaction mixture. This means that if the user wantsthe reaction mixture to remain within 1° C. of 94° C. for 5 seconds inthe denaturation segment, and programs in these parameters, he or shegets the 5 second denaturation interval with less time lag than withconventional tubes with thicker walls. This performance characteristicof being able to program a short soak interval such as a 5 seconddenaturation soak and get a soak at the programmed temperature for theexact programmed time is enabled by use of a calculated sampletemperature to control the timer. In the system described herein, thetimer to time an incubation or soak interval is not started until thecalculated sample temperature reaches the programmed soak temperature.

Further, with the thin walled sample tubes, it only takes about one-halfto two-thirds as long for the sample mixture to get within 1° C. of thetarget temperature as with prior art thick-walled microcentrifuge tubesand this is true both with the tall MAXIAMP™ tube shown in FIG. 50 andthe smaller thin walled MICROAMP™ tube shown in FIG. 15.

The wall thickness of both the MAXIAMP™ and MICROAMP™ tubes iscontrolled tightly in the manufacturing process to be as thin aspossible consistent with adequate structural strength. Typically, forpolypropylene, this will be anywhere from 0.009 to 0.012 inches. If new,more exotic materials which are stronger than polypropylene are used toachieve the advantage of speeding up the PCR reaction, the wallthickness can be less so long as adequate strength is maintained towithstand the downward force to assure good thermal contact, and otherstresses of normal use. With a height (dimension B in FIG. 50) of 1.12inches and a dimension C of 0.780 inches, the MAXIAMP tube's timeconstant is approximately 14 seconds although this has not beenprecisely measured as of the time of filing. The MICROAMP tube timeconstant for the shorter tube shown in FIG. 15 is typicallyapproximately 9.5 seconds with a tube wall thickness in the conicalsection of 0.009 inches plus or minus 0.001 inches.

FIG. 51 shows the results of use of the thinner walled MICROAMP tube. Asimilar speeded up attainment of target temperatures will result fromuse of the thin walled MAXIAMP tube.

Referring to FIG. 51, there is shown a graph of the relative times forthe calculated sample temperature in a MICROAMP tube versus the time fora prior art tube to reach a temperature within 1° C. of a targetdenaturation temperature of 94° C from a starting temperature of 72° C.In FIG. 51, a 100 microliter sample was present in each tube. The curvewith data points marked by open boxes is the calculated sampletemperature response for a MICROAMP tube with a 9.5 second response timeand a 0.009 inch wall thickness. The curve with data points marked byX's represents the calculated sample temperature for a 100 microlitersample in a prior art, thick walled microcentrifuge tube with a 0.030inch wall thickness. This graph shows that the thin walled MICROAMP tubesample reaches a calculated temperature within 1° C. of the94° C. targetsoak temperature within approximately 36 seconds while the prior arttubes take about 73 seconds. This is important because in instrumentswhich do not start their timers until the soak temperature issubstantially achieved, the prior art tubes can substantially increaseoverall processing time especially when considered in light of the factthat each PCR cycle will have at least two ramps and soaks and there aregenerally very many cycles performed. Doubling the ramp time for eachramp by using prior art tubes can therefore drastically increaseprocessing time. In systems which start their times based uponblock/bath/oven temperature without regard to actual sample temperature,these long delays between changes in block/bath/oven temperature andcorresponding changes in sample mixture temperature can have seriousnegative consequences. The problem is that the long delay can cut intothe time that the reaction mixture is actually at the temperatureprogrammed for a soak. For very short soaks as are popular in the latestPCR processes, the reaction mixture may never actually reach theprogrammed soak temperature before the heating/cooling system startsattempting to change the reaction mixture temperature.

FIG. 50 shows a polypropylene cap 650 connected to the MAXIAMP sampletube by a plastic web 652. The outside diameter E of the cap and theinside diameter F of the tube upper section are sized for aninterference fit of between 0.002 and 0.005 inches. The thickness of thewall and dome portions of the cap in a preferred embodiment isapproximately 0.020 inches. The inside surface 654 of the tube should befree of flash, nicks and scratches so that a gas-tight seal with the capcan be formed.

FIG. 52 shows a plan view of the tube 651, the cap 650 and the web 652.A shoulder 656 prevents the cap from being pushed too far down into thetube and allows sufficient projection of the cap above the top edge ofthe sample tube for making contact with the heated platen. This alsoallows sufficient cap deformation such that the minimum acceptable forceF in FIG. 15 can be applied by deformation of the cap.

In the preferred embodiment, the tube and cap are made of Himont PD701polypropylene which is autoclavable at temperatures up to 126° C. fortimes up to 15 minutes. This allows the disposable tubes to besterilized before use. Since the caps are permanently deformed in use inmachines with heated platens, the tubes are designed for use only once.

Caps for the MICROAMP tubes are available in connected strips of 8 or 12caps with each cap numbered or as individual caps. Single rows of capsmay be used and the rows may be easily shortened to as few as desired orindividual caps may be cut off the strip. Caps for MAXIAMP tubes areeither attached as shown in FIG. 50, or are separate individual caps.

The maximum volume for post-PCR reagent additions to permit mixing onthe MICROAMP tube is 200 microliters and is up to 500 microliters forthe MAXIAMP tube. Temperature limits are -70° C. to 126° C.

The response time depends upon the volume of the sample. Response ismeasured as the time for the sample to come within 37% of the newtemperature when the block suddenly changes temperature. Typicalresponse time for a 50 microliter fill are 7.0 seconds and for a 20microliter fill are 5.0 seconds.

Electronics and Software Version 1

Referring to FIGS. 47A and 47B (hereafter FIG. 47), there is shown ablock diagram for the electronics of a preferred embodiment of a controlsystem in a class of control systems represented by CPU block 10 inFIG. 1. The purpose of the control electronics of FIG. 47 is, interalia, to receive and store user input data defining the desired PCRprotocol, read the various temperature sensors, calculate the sampletemperature, compare the calculated sample temperature to the desiredtemperature as defined by the user defined PCR protocol, monitor thepower line voltage and control the film heater zones and the rampcooling valves to carry out the desired temperature profile of the userdefined PCR protocol.

A microprocessor (hereafter CPU) 450 executes the control programdescribed below and given in Microfiche Appendix C in source code form.In the preferred embodiment, the CPU 450 is an OKI CMOS 8085. The CPUdrives an address bus 452 by which various ones of the other circuitelements in FIG. 47 are addressed. The CPU also drives a data bus 454 bywhich data is transmitted to various of the other circuit elements inFIG. 47.

The control program of Microfiche Appendix C and some system constantsare stored in EPROM 456. User entered data and other system constantsand characteristics measured during the install process (install programexecution described below) are stored in battery backed up RAM 458. Asystem clock/calendar 460 supplies the CPU 450 with date and timeinformation for purposes of recording a history of events during PCRruns and the duration of power failures as described below in thedescription of the control software.

An address decoder 462 receives and decodes addresses from the addressbus 452 and activates the appropriate chip select lines on a chip selectbus 464.

The user enters PCR protocol data via a keyboard 466 in response toinformation displayed by CPU on display 468. The two way communicationbetween the user and the CPU 450 is described in more detail below inthe user interface section of the description of the control software. Akeyboard interface circuit 470 converts user keystrokes to data which isread by the CPU via the data bus 454.

Two programmable interval timers 472 and 474 each contain counters whichare loaded with counts calculated by the CPU 450 to control theintervals during which power is applied to the various film heaterzones.

An interrupt controller 476 sends interrupt requests to the CPU 450every 200 milliseconds causing the CPU 450 to run the PID task describedbelow in the description of the control software. This task reads thetemperature sensors and calculates the heating or cooling powernecessary to move the sample temperature from its current level to thelevel desired by the user for that point in time in the PCR protocolbeing executed.

A UART 478 services an RS232 interface circuit 480 such that data storedin the RAM 480 may be output to a printer. The control softwaremaintains a record of each PCR run which is performed with respect tothe actual temperatures which existed at various times during the runfor purposes of user validation that the PCR protocol actually executedcorresponded to the PCR protocol desired by the user. In addition, userentered data defining the specific times and temperatures desired duringa particular PCR protocol is also stored. All this data and other dataas well may be read by the CPU 450 and output to a printer coupled tothe RS232 port via the UART 478. The RS232 interface also allows anexternal computer to take control of the address and data buses forpurposes of testing.

A peripheral interface chip (hereafter PIC) 482 serves as a programmableset of 4 input/output registers. At power-up, the CPU 450 selects thePIC 482 via the address decoder 462 and the chip select bus 464. The CPUthen writes a data word to the PIC via data bus 454 to program the PIC482 regarding which registers are to be output ports and which are to beinput ports. Subsequently, the CPU 450 uses the output registers tostore data words written therein by the CPU via the data bus 454 tocontrol the internal logic state of a programmable array logic chip(PAL) 484.

The PAL 484 is a state machine which has a plurality of input signalsand a plurality of output signals. PAL's in general contain an array oflogic which has a number of different states. Each state is defined bythe array or vector of logic states at the inputs and each state resultsin a different array or vector of logic states on the outputs. The CPU450, PIC 482, PAL 484 and several other circuits to be defined belowcooperate to generate different states of the various output signalsfrom the PAL 484. These different states and associated output signalsare what control the operation of the electronics shown in FIG. 47 aswill be described below.

A 12 bit analog-to-digital converter (A/D) 486 converts analog voltageson lines 488 and 490 to digital signals on data bus 454. These are readby the CPU by generating an address for the A/D converter such that achip select signal on bus 464 coupled to the chip select input of theA/D converter goes active and activates the converter. The analogsignals on lines 488 and 490 are the output lines of two multiplexers492 and 494. Multiplexer 492 has four inputs ports, each having twosignal lines. Each of these ports is coupled to one of the fourtemperature sensors in the system. The first port is coupled to thesample block temperature sensor. The second and third ports are coupledto the coolant and ambient temperature sensors, respectively and thefourth port is coupled to the heated cover temperature sensor. A typicalcircuit for each one of these temperature sensors is shown in FIG. 48. A20,000 ohm resistor 496 receives at a node 497 a regulated +15 voltregulated power supply 498 in FIG. 47 via a bus connection line which isnot shown. This + 15 volts D.C. signal reverse biases a zener diode 500.The reverse bias current and the voltage drop across the zener diode arefunctions of the temperature. The voltage drop across the diode is inputto the multiplexer 292 via lines 502 and 504. Each temperature sensorhas a similar connection to the multiplexer 292.

Multiplexer 494 also has 4 input ports but only three are connected. Thefirst input port is coupled to a calibration voltage generator 506. Thisvoltage generator outputs two precisely controlled voltage levels to themultiplexer inputs and is very thermally stable. That is, the referencevoltage output by voltage source 506 drifts very little if at all withtemperature. This voltage is read from time to time by the CPU 450 andcompared to a stored constant which represents the level this referencevoltage had at a known temperature as measured during execution of theinstall process described below. If the reference voltage has driftedfrom the level measured and stored during the install process, the CPU450 knows that the other electronic circuitry used for sensing thevarious temperatures and line voltages has also drifted and adjuststheir outputs accordingly to maintain very accurate control over thetemperature measuring process.

The other input to the multiplexer 494 is coupled via line 510 to anRMS-to-DC converter circuit 512. This circuit has an input 514 coupledto a step-down transformer 516 and receives an A.C. voltage at input 514which is proportional to the then existing line voltage at A.C. powerinput 518. The RMS-to-DC converter 512 rectifies the A.C. voltage andaverages it to develop a D.C. voltage on line 510 which also isproportional to the A.C. input voltage on line 518.

Four optically coupled triac drivers 530, 532, 534 and 536 receive inputcontrol signals via control bus 538 from PAL logic 484. Each of thetriac drivers 530, 532 and 534 controls power to one of the three filmheater zones. These heater zones are represented by blocks 254, 260/262and 256/258 (the same reference numerals used in FIG. 13). The triacdriver 536 controls power to the heated cover, represented by block 544via a thermal cut-out switch 546. The heater zones of the film heaterare protected by a block thermal cutout switch 548. The purpose of thethermal cutout switches is to prevent meltdown of the film heater/sampleblock on the heated cover in case of a failure leading to the triacdrivers being left on for an unsafe interval. If such an event happens,the thermal cut-out switches detect an overly hot condition, and shutdown the triacs via signals on lines 552 or 554.

The main heater zone of the film heater is rated at 360 watts while themanifold and edge heater zones are rated at 180 watts and 170 wattsrespectively. The triac drivers are Motorola MAC 15A10 15 amp triacs.Each heater zone is split into 2 electrically isolated sections eachdissipating 1/2 the power. The 2 halves are connected in parallel forline voltages at 518 less than 150 volts RMS. For line voltages greaterthan this, the two halves are connected in series. These alternateconnections are accomplished through a "personality" plug 550.

The AC power supply for the film heater zones is line 559, and the ACsupply for the heated cover is via line 560.

A zero crossing detector 566 provides basic system timing by emitting apulse on line 568 at each zero crossing of the AC power on line 518. Thezero crossing detector is a National LM 311N referenced to analog groundand has 25 mV of hysteresis. The zero crossing detector takes its inputfrom transformer 516 which outputs A.C. signal from 0 to 5.52 volts foran A.C. input signal of from 0 to 240 volts A.C.

A power transformer 570 supplies A.C. power to the pump 41 that pumpscoolant through the ramp and bias cooling channels. The refrigerationunit 40 also receives its A.C. power from the transformer 570 viaanother portion of the personality plug 550. The transformer 550 alsosupplies power to three regulated power supplies 572, 498 and 574 andone unregulated power supply 576.

For accuracy purposes in measuring the temperatures, the calibrationvoltage generator 506 uses a series of very precise, thin-film, ultralowtemperature drift 20K ohm resistors (not shown in FIG. 47 but shown asresistors RA1 in the schematics of Microfiche Appendix E). These sameultralow drift resistors are used to set the gain of an analog amplifier578 which amplifies the output voltage from the selected temperaturesensor prior to conversion to a digital value. These resistors driftonly 5 ppm/C°.

All the temperature sensors are calibrated by placing them (separatedfrom the structures whose temperatures they measure) first in a stable,stirred-oil, temperature controlled bath at 40° C. and measuring theactual output voltages at the inputs to the multiplexer 492. Thetemperature sensors are then placed in a bath at a temperature of 95° C.and their output voltages are again measured at the same points. Theoutput voltage of the calibration voltage generator 506 is also measuredat the input of the multiplexer 494. For each temperature, the digitaloutput difference from the A/D converter 486 between each of thetemperature sensor outputs and the digital output that results from thevoltage generated by the calibration voltage generator 506 is measured.The calibration constants for each temperature sensor to calibrate eachfor changes in temperature may then be calculated.

The sample block temperature sensor is then subjected to a furthercalibration procedure. This procedure involves driving the sample blockto two different temperatures. At each temperature level, the actualtemperature of the block in 16 different sample wells is measured using16 RTD thermocouple probes accurate to within 0.02° C. An averageprofile for the temperature of the block is then generated and theoutput of the A/D converter 464 is measured with the block temperaturesensor in its place in the sample block. This is done at bothtemperature levels. From the actual block temperature as measured by theRTD probes and the A/D output for the block temperature sensor, afurther calibration factor can be calculated. The temperaturecalibration factors so generated are stored in battery backed up RAM458. Once these calibration factors are determined for the system, it isimportant that the system not drift appreciably from the electricalcharacteristics that existed at the time of calibration. It is importanttherefore that low drift circuits be selected and that ultralow driftresistors be used. The selections made for the analog components for anexemplary embodiment are given in Microfiche Appendix E.

The manner in which the CPU 450 controls the sample block temperaturecan be best understood by reference to the section below describing thecontrol program. However, to illustrate how the electronic circuitry ofFIG. 47 cooperates with the control software to carry out a PCR protocolconsider the following.

The zero crossing detector 566 has two outputs in output bus 568. One ofthese outputs emits a negative going pulse for every positive goingtransition of the A.C. signal across the zero voltage reference. Theother emits a negative pulse upon every negative-going transition of theA.C. signal across the zero reference voltage level. These two pulses,shown typically at 580 define one complete cycle or two half cycles. Itis the pulse trains on bus 568 which define the 200 millisecond sampleperiods. For 60 cycle/sec A.C. as found in the U.S., 200 millisecondscontains 24 half cycles.

A typical sample period is shown in FIG. 49. Each "tick" mark in FIG. 49represents one half cycle. During each 200 msec sample period, the CPU450 is calculating the amount of heating or cooling power needed tomaintain the sample block temperature at a user defined setpoint orincubation temperature or to move the block temperature to a newtemperature depending upon where in the PCR protocol time line theparticular sample period lies. The amount of power needed in each filmheater zone is converted into a number of half cycles each heater zoneis to remain off during the next 200 msec sample period. Just before theend of the current sample period in which these calculations are made,the CPU 450 addresses each of the 4 timers in the programmable intervaltimer (PIT) 472. To each timer, the CPU writes data constituting a"present" count representing the number of half cycles the heater zoneassociated with that timer is to remain off in the next sample period.In FIG. 49, this data is written to the timers during interval 590 justpreceding the starting time 592 of the next sample period. Assume that arapid ramp up to the denaturation temperature of 94° C. is called for bythe user setpoint data for an interval which includes the sampleinterval between times 592 and 594. Accordingly, the film heaters willbe on for most of the period. Assume that the central zone heater is tobe on for all but three of the half cycles during the sample period. Inthis case, the CPU 450 writes a three into the counter in PIT 472associated with the central zone heater during interval 590. This writeoperation automatically causes the timer to issue a "shut off" signal onthe particular control line of bus 592 which controls the central zoneheater. This "shut off" signal causes the PAL 484 to issue a "shut off"signal on the particular one of the signal lines in bus 538 associatedwith the central zone. The triac driver 530 then shuts off at the nextzero crossing, i.e., at time 592. The PIT receives a pulse train ofpositive-going pulses on line 594 from the PAL 484. These pulses aretranslations of the zerocrossing pulses on 2-line bus 568 by PAL 484into positive going pulses at all zero crossing pulses on 2-line bus 568by PAL 484 into positive going pulses at all zero crossings on a singleline, i.e., line 594. The timer in PIT 472 associated with the centralfilm heater zone starts counting down from its present count of 3 usingthe half cycle marking pulses on line 594 as its clock. At the end ofthe third half cycle, this timer reaches 0 and causes its output signalline on bus 592 to change states. This transition from the off to onstate is shown at 596 in FIG. 49. This transition is communicated to PAL484 and causes it to change the state of the appropriate output signalon bus 538 to switch the triac driver 530 on at the third zero-crossing.Note that by switching the triacs on at the zero crossings as is done inthe preferred embodiment, switching off of a high current flowingthrough an inductor (the film heater conductor) is avoided. Thisminimizes the generation of radio frequency interference or other noise.Note that the technique of switching a portion of each half cycle to thefilm heater in accordance with the calculated amount of power neededwill also work as an alternative embodiment, but is not preferredbecause of the noise generated by this technique.

The other timers of PIT 472 and 474 work in a similar manner to managethe power applied to the other heater zones and to the heated cover inaccordance with power calculated by the CPU.

Ramp cooling is controlled by CPU 450 directly through the peripheralinterface 482. When the heating/cooling power calculations performedduring each sample period indicate that ramp cooling power is needed,the CPU 450 addresses the peripheral interface controller (PIC) 482. Adata word is then written into the appropriate register to drive outputline 600 high. This output line triggers a pair of monostablemultivibrators 602 and 604 and causes each to emit a single pulse, onlines 606 and 608, respectively. These pulses each have peak currentsjust under 1 ampere and a pulse duration of approximately 100milliseconds. The purpose of these pulses is to drive the solenoid valvecoils that control flow through the ramp cooling channels very hard toturn on ramp cooling flow quickly. The pulse on line 606 causes a driver610 to ground a line 612 coupled to one side of the solenoid coil 614 ofone of the solenoid operated valves. The other terminal of the coil 614is coupled to a power supply "rail" 616 at +24 volts DC from powersupply 576. The one shot 602 controls the ramp cooling solenoid operatedvalve for flow in one direction, and the one shot 604 controls thesolenoid operated valve for flow in the opposite direction.

Simultaneously, the activation of the RCOOL signal on line 600 causes adriver 618 to be activated. This driver grounds the line 612 through acurrent limiting resistor 620. The value of this current limitingresistor is such that the current flowing through line 622 is at leastequal to the hold current necessary to keep the solenoid valve 614 open.Solenoid coils have transient characteristics that require largecurrents to turn on a solenoid operated valve but substantially lesscurrent to keep the valve open. When the 100 msec pulse on line 606subsides, the driver 612 ceases directly grounding the line 612 leavingonly the ground connection through the resistor 620 and driver 618 forholding current.

The solenoid valve 614 controls the flow of ramp cooling coolant throughthe sample block in only 1/2 the ramp cooling tubes, i.e., the tubescarrying the coolant in one direction through the sample block. Anothersolenoid operated valve 624 controls the coolant flow of coolant throughthe sample block in the opposite direction. This valve 624 is driven inexactly the same way as solenoid operated valve 614 by drivers 626 and628, one shot 604 and line 608.

The need for ramp cooling is evaluated once every sample period. Whenthe PID task of the control software determines from measuring the blocktemperature and comparing it to the desired block temperature that rampcooling is no longer needed, the RCOOL signal on line 600 isdeactivated. This is done by the CPU 450 by addressing the PIC 482 andwriting data to it which reverses the state of the appropriate bit inthe register in PIC 482 which is coupled to line 600.

The logic equations for PAL 484 are attached hereto as MicroficheAppendix D. The logic equations for the address decoder 462, which isalso programmable array logic, are also attached hereto is MicroficheAppendix D.

The PIT 474 also has two other timers therein which time a 20 Hzinterrupt and a heating LED which gives a visible indication when thesample block is hot and unsafe to touch.

The system also includes a beeper one shot 630 and a beeper 632 to warnthe user when an incorrect keystroke has been made.

The programmable interrupt controller 476 is used to detect 7interrupts; Level 1-test; Level 2-20 Hz; Level 3 - Transmit Ready; Level4 - Receive ready; Level 5 - Keyboard interrupt; Level 6 - Main heaterturn on; and, Level 7 - A.C. line zero cross.

The programmable peripheral interface 482 has four outputs (not shown)for controlling the multiplexers 492 and 494. These signals MUX1 EN andMUX2 EN enable one or the other of the two multiplexers 492 and 494while the signals MUX 0 and MUX 1 control which channel is selected forinput to the amplifier 578. These signals are managed so that only onechannel from the two multiplexers can be selected at any one time.

An RLTRIG* signal resets a timeout one shot 632 for the heaters whichdisables the heaters via activation of the signal TIMEOUT EN* to the PAL484 if the CPU crashes. That is, the one shot 632 has a predeterminedinterval which it will wait after each reset before it activates thesignal TIMEOUT EN* which disables all the heater zones. The CPU 450executes a routine periodically which addresses the PIC 482 and writesdata to the appropriate register to cause activation of a signal on line634 to reset the one shot 632. If the CPU 450 "crashes" for any reasonand does not execute this routine, the timeout one-shot 632 disables allthe heater zones.

The PIC 482 also has outputs COVHTR EN* and BLKHTREN* (not shown) forenabling the heated cover and the sample block heater. Both of thesesignals are active low and are controlled by the CPU 450. They areoutput to the PAL 484 via bus 636.

The PIC 482 also outputs the signals BEEP and BEEPCLR* on bus 640 tocontrol the beeper one shot 630.

The PIC 482 also outputs a signal MEM1 (not shown) which is used toswitch pages between the high address section of EPROM 456 and the lowaddress section of battery RAM 458. Two other signals PAGE SEL 0 andPAGE SEL 1 (not shown) are output to select between four 16K pages inEPROM 456.

The four temperature sensors are National LM 135 zener diode typesensors with a zener voltage/temperature dependence of 10 mV/° K. Thezener diodes are driven from the regulated power supply 498 through the20K resistor 496. The current through the zeners varies fromapproximately 560 μA to 615 μA over the 0° C. to 100° C. operatingrange. The zener self heating varies from 1.68 mW to 2.10 mW over thesame range.

The multiplexers 492 and 494 are DG409 analog switches. The voltages onlines 488 and 490 are amplified by an AD625KN instrumentation amplifierwith a transfer function of V_(OUT) =3*V_(IN) -7.5. The A/D converter486 is an AD7672 with an input range from 0-5 volts. With the zenertemperature sensor output from 2.73 to 3.73 volts over the 0° C. to 100°C. range, the output of the amplifier 578 will be 0.69 volts to 3.69volts, which is comfortably within the A/D input range.

The keys to highly accurate system performance are good accuracy and lowdrift with changes in ambient temperature. Both of these goals areachieved by using a precision voltage reference source, i.e.,calibration voltage generator 506, and continuously monitoring itsoutput through the same chain of electronics as are used to monitor theoutputs of the temperature sensors and the AC line voltage on line 510.

The calibration voltage generator 506 outputs two precision voltages onlines 650 and 652. One voltage is 3.75 volts and the other is 3.125volts. These voltages are obtained by dividing down a regulated supplyvoltage using a string of ultralow drift, integrated, thin filmresistors with a 0.05% match between resistors and a 5 ppm/degree C.temperature drift coefficient between resistors. The calibration voltagegenerator also generates -5 volts for the A/D converter referencevoltage and 7.5 volts for the instrumentation amplifier offset. Thesetwo voltages are communicated to the A/D 486 and the amplifier 578 bylines which are not shown. These two negative voltages are generatedusing the same thin film resistor network and OP 27 GZ op-amps (notshown). The gain setting resistors for the operational amplifier 578 arealso the ultralow drift, thin-film, integrated, matched resistors.

The control firmware, control electronics and the block design aredesigned such that well-to-well and instrument-to-instrumenttransportability of PCR protocols is possible.

High throughput laboratories benefit from instruments which are easy touse for a wide spectrum of lab personnel and which require a minimalamount of training. The software for the invention was developed tohandle complex PCR thermocycling protocols while remaining easy toprogram. In addition, it is provided with safeguards to assure theintegrity of samples during power interruptions, and can document thedetailed events of each run in safe memory.

After completing power-up self-checks shown in FIGS. 53 and 54, toassure the operator that the system is operating properly, the userinterface of the invention offers a simple, top-level menu, inviting theuser to run, create or edit a file, or to access a utility function. Noprogramming skills are required, since pre-existing default files can bequickly edited with customized times and temperatures, then stored inmemory for later use. A file protection scheme prevents unauthorizedchanges to any user's programs. A file normally consists of a set ofinstructions to hold a desired temperature or to thermocycle. Complexprograms are created by linking files together to form a method. Acommonly used file, such as a 4° C. incubation following a thermocycle,can be stored and then incorporated into methods created by other users.A new type of file, the AUTO file is a PCR cycling program which allowsthe user to specify which of several types of changes to controlparameters will occur each cycle: time incrementing (auto segmentextension, for yield enhancement), time decrementing, or temperatureincrementing or decrementing. For the highest degree of controlprecision and most reliable methods transferability, temperatures aresetable to 0.1° C., and times are programmed to the nearest second. Theinvention has the ability to program a scheduled PAUSE at one or moresetpoints during a run for reagent additions or for removal of tubes atspecific cycles.

The system of the invention has the ability to store a 500 recordhistory file for each run. This feature allows the user to review theindividual steps in each cycle and to flag any special status or errormessages relating to irregularities. With the optional printer, theinvention provides hardcopy documentation of file and method parameters,run-time time/temperature data with a time/date stamp, configurationparameters, and sorted file directories.

In order to assure reproducible thermocycling, the computed sampletemperature is displayed during the ramp and hold segments of eachcycle. A temperature one degree different than the set temperature isnormally used to trigger the ramp-time and hold-time clocks, but thiscan be altered by the user. Provided the proper time constant for thetype of tube and volume is used (described more fully elsewhere herein),the sample will always approach the desired sample temperature with thesame accuracy, regardless of whether long or short sample incubationtimes have been programmed. Users can program slow ramps for thespecialized annealing requirements of degenerate primer pools, or veryshort (1-5 sec) high-temperature denaturation periods for very GC richtargets. Intelligent defaults are preprogrammed for 2 - and3-temperature PCR cycles.

Diagnostic tests can be accessed by any users to check the heating andcooling system status, since the software gives Pass/Fail reports. Inaddition, a system performance program performs a comprehensivesubsystem evaluation and generates a summary status report.

The control firmware is comprised of several sections which are listedbelow:

Diagnostics

Calibration

Install

Real time operating system

Nine prioritized tasks that manage the system

Start-up sequence

User interface

The various sections of the firmware will be described with eithertextual description, pseudocode or both. The actual source code in Clanguage is included below as Microfiche Appendix C.

Features of the firmware are:

1. A Control system that manages the average sample block temperature towithin ±0.1° C. as well as maintaining the temperature non-uniformity asbetween wells in the sample block to within ±0.5° C.

2. A temperature control system that measures and compensates for linevoltage fluctuations and electronic temperature drift.

3. Extensive power up diagnostics that determine if system componentsare working.

4. Comprehensive diagnostics in the install program which qualify theheating and cooling systems to insure they are working properly.

5. A logical and organized user interface, employing a menu drivensystem that allows instrument operation with minimal dependency on theoperators manual.

6. The ability to link up to 17 PCR protocols and store them as amethod.

7. The ability to store up to 150 PCR protocols and methods in the userinterface.

8. A history file that records up to 500 events of the previous run aspart of the sequence task.

9. The ability to define the reaction volume and tube size type at thestart of a run for maximum temperature accuracy and control as part ofthe user interface and which modifies tau (the tube time constant) inthe PID task.

10. Upon recovery from a power failure, the system drives the sampleblock to 4° C. to save any samples that may be loaded in the samplecompartment. The analyzer also reports the duration of the power failureas part of the start-up sequence.

11. The ability to print history file contents, "run time" parametersand stored PCR protocol parameters as part of the print task.

12. The ability to configure the temperature to which the apparatus willreturn during any idle state.

13. The ability to check that the setpoint temperature is reached withina reasonable amount of time.

14. The ability to control the instrument remotely via an RS232 port.

There are several levels of diagnostics which are described below:

A series of power-up tests are automatically performed each time theinstrument is turned on. They evaluate critical areas of the hardwarewithout user intervention. Any test that detects a component failurewill be run again. If the test fails twice, an error message isdisplayed and the keyboard is electronically locked to prevent the userfrom continuing.

The following areas are tested:

Programmable Peripheral Interface device

Battery RAM device

Battery RAM checksum

EPROM devices

Programmable Interface Timer devices

Clock/Calendar device

Programmable Interrupt Controller device

Analog to Digital section

Temperature sensors

Verify proper configuration plug

A Series of service only diagnostics are available to final testers atthe manufacturer's location or to field service engineers through a"hidden" keystroke sequence (i.e. unknown to the customer). Many of thetests are the same as the ones in the start up diagnostics with theexception that they can be continually executed up to 99 times.

The following areas are tested:

Programmable Peripheral Interface device

Battery RAM device

Battery RAM checksum

EPROM devices

Programmable Interface Timer devices

Clock/Calendar device

Programmable Interrupt Controller device

Analog to Digital section

RS-232 section

Display section

Keyboard

Beeper

Ramp Cooling Valves

Check for EPROM mismatch

Firmware version level

Battery RAM Checksum and Initialization

Autostart Program Flag

Clear Calibration Flag

Heated Cover heater and control circuitry

Edge heater and control circuitry

Manifold heater and control circuitry

Central heater and control circuitry

Sample block thermal cutoff test

Heated cover thermal cutoff test

User diagnostics are also available to allow the user to perform a quickcool and heat ramp verification test, an extensive confirmation of theheating and cooling system. These diagnostics also allow the user toview the history file, which is a sequential record of events thatoccurred in the previous run. The records contain time, temperature,setpoint number, cycle number, program number and status messages.

Remote Diagnostics are available to allow control of the system from anexternal computer via the RS-232 port. Control is limited to the servicediagnostics and instrument calibration only.

Calibration to determine various parameters such as heater resistance,etc. is performed. Access to the calibration screen is limited by a"hidden" key sequence (i.e. unknown to the customer). The followingparameters are calibrated:

The configuration plug is a module that rewires the chiller unit, sampleblock heaters, coolant pump and power supplies for the proper voltageand frequency (100V/50 Hz, 100/60 Hz, 120/60 Hz, 220/50 Hz or 230/50Hz). The user enters the type of configuration plug installed. Thefirmware uses this information to compute the equivalent resistance ofthe sample block heaters. Upon power-up, the system verifies that theconfiguration plug selected is consistent with the current line voltageand frequency.

The heater resistance must be determined in the calibration process sothat precise calculations of heater power delivered can be made. Theuser enters the actual resistances of the six sample block heaters (twomain heaters, two manifold heaters and two edge heaters). Theconfiguration plug physically wires the heater in series for 220-230 VACand in parallel for 100-120 VAC operation. The firmware computes theequivalent resistance of each of the three heaters by the followingformula:

    For 100-120 VAC: R.sub.eq =(R.sub.1 * R.sub.2)/R.sub.1 +R.sub.2 (7)

    For 220-230 VAC: R.sub.eq =R.sub.1 +R.sub.2                (8)

The equivalent resistance is used to deliver a precise amount of heatingpower to the sample block (Power=Voltage² ×Resistance).

The calibration of the A/D circuit is necessary so that temperatures canbe precisely measured. This is performed by measuring two test pointvoltages (TP6 and TP7 on the CPU board) and entering the measuredvoltages. The output of the A/D at each voltage forms the basis of a twopoint calibration curve. These voltages are derived from a 5 voltprecision source and are accurate and temperature independent. At thestart of each run, these voltages are read by the system to measureelectronic drift due to temperature because any changes in A/D output isdue to temperature dependencies in the analog chain (multiplexer, analogamplifier and A/D converter).

Calibration of the four temperature sensors (sample block, ambient,coolant and heated cover) is performed for accurate temperaturemeasurements. Prior to installation into an instrument, the ambient,coolant, and heated cover temperature sensors are placed in a water bathwhere their output is recorded (XX.X° C. at YYYY mV). These values arethen entered into the system. Since temperature accuracy in these areasis not critical, a one point calibration curve is used.

The sample block sensor is calibrated in the instrument. An array of 15accurate temperature probes is strategically placed in the sample blockin the preferred embodiment. The output of the temperature probes iscollected and averaged by a computer. The firmware commands the block togo to 40° C. After a brief stabilizing period the user enters theaverage block temperature as read by the 15 probes. This procedure isrepeated at 95° C. forming a two point calibration curve.

Calibration of the AC to DC line voltage sampling circuit is performedby entering into the system the output of the AC to DC circuit for twogiven AC input voltages, forming a two point calibration curve. Theoutput of the circuit is not linear over the required range (90-260 VAC)and therefore requires two points at each end (100 and 120, 220 and 240VAC), but only uses one set based on the current input voltage.

An accurate measure of AC voltage is necessary to deliver a preciseamount of power to the sample block (Power=Voltage² ×Resistance). TheInstall program is a diagnostic tool that performs an extensive test ofthe cooling and heating systems. Install measures or calculates controlcooling conductance, ramp cooling conductance at 10° C. and 18° C.,cooling power at 10° C. and 20° C., sample block thermal and coolantcapacity and sample block sensor lag. The purpose of install is threefold:

1. To uncover marginal or faulty components.

2. To use some of the measured values as system constants stored inbattery backed up RAM to optimize the control system for a giveninstrument.

3. To measure heating and cooling system degradation over time.

Install is executed once before the system is shipped and should also berun before use or whenever a major component is replaced. The Installprogram may also be run by the user under the user diagnostics.

The heater ping test verifies that the heaters are properly configuredfor the current line voltage (i.e. in parallel for 90-132 VAC and inseries for 208-264 VAC). The firmware supplies a burst of power to thesample block and then monitors the rise in temperature over a

10 second time period. If the temperature rise is outside a specifiedramp rate window, then the heaters are incorrectly wired for the currentline voltage and the install process is terminated.

The control cooling conductance tests measures the thermal conductanceK_(cc) across the sample block to the control cooling passages. Thistest is performed by first driving the sample block temperature to 60°C. (ramp valves are closed), then integrating the heater power requiredto maintain the block at 60° C. over a 30 second time period. Theintegrated power is divided by the sum of the difference between theblock and coolant temperature over the interval.

    K.sub.cc =Σ Heater Power.sub.60° C. /Σ Block-Coolant Temp                                                      (9)

Typical values are 1.40 to 1.55 Watts/C. A low K_(cc) may indicate aclogged liner(s). A high K_(cc) may be due to a ramp valve that is notcompletely closed, leakage of the coolant to the outside diameter of theliner, or a liner that has shifted.

The block thermal capacity (Blk Cp) test measures the thermal capacityof the sample block by first controlling the block at 35° C. thenapplying the maximum power to the heaters for 20 seconds. The blockthermal capacity is equal to the integrated power divided by thedifference in block temperature. To increase accuracy, the effect ofbias cooling power is subtracted from the integrated power.

    Blk Cp=ramp time * (heater-control cool pwr)/ delta temp.  (10)

where:

ramp time=20 seconds

heater power=500 watts

control cool=(Σ block-coolant temp) * K_(cc)

delta temp=TBlock_(t=20) -TBlock_(t=0)

The typical value of Block Cp is 540 watt-seconds/°C.±30. Assuming anormal K_(cc) value, an increase in block thermal capacity is due to anincrease in thermal loads, such as moisture in the foam backing, loss ofinsulation around the sample block, or a decrease in heater power suchas a failure of one of the six heater zones or a failure of theelectronic circuitry that drives the heater zones, or an incorrect or anincorrectly wired voltage configuration module.

A chiller test measures the system cooling output in watts at 10° C. and18° C. The system cooling power, or chiller output, at a giventemperature is equal to the summation of thermal loads at thattemperature. The main components are: 1. heating power required tomaintain the block at a given temperature, 2. power dissipated by thepump used to circulate the coolant around the system, and 3. losses inthe coolant lines to the ambient. The chiller power parameter ismeasured by controlling the coolant temperature at either 10° C. or 18°C. and integrating the power applied to the sample block to maintain aconstant coolant temperature, over a 32 second interval. The differencebetween the block and coolant temperature is also integrated to computelosses to ambient temperature.

    Chiller power=Σ Heating power+Pump power+(Kamb * Σ (blk-cool temp))                                                    (11)

where:

heating power=Sum of heating power required to maintain coolant at 10°C. or 18° C. over time 32 seconds.

Pump Power=Circulating pump, 12 watts

Kamb=Conductance to ambient, 20 watts/°C.

blk-cool temp=Sum of difference in block and coolant temp over time 32seconds

The typical value for chiller power is 230 watts±40 at C. and 370watts±30 at 18° C. Low chiller power may be due to an obstruction in thefan path, a defective fan, or a marginal or faulty chiller unit. It mayalso be due to a miswired voltage configuration plug.

A ramp cooling conductance (K_(c)) test measures the thermal conductanceat 10° C. and 18° C. across the sample block to the ramp and controlcooling passages. This test is performed by first controlling thecoolant temperature at 10° C. or 18° C., then integrating, over a 30second time interval, the heating power applied to maintain the coolantat the given temperature divided by the difference of block and coolanttemperature over the time interval.

    K.sub.c =Σ Heating power/Σ (block-coolant temperature)(12)

Typical values for K_(c) are 28 watts/° C. ±3 at 10° C. and 31 watts/°C.±3 at 18° C. A low K_(c) may be due to a closed or obstructed rampvalve, kinked coolant tubing, weak pump or a hard water/Prestone™mixture.

A sensor lag test measures the block sensor lag by first controlling theblock temperature to 35° C. and then applying 500 watts of heater powerfor 2 seconds and measuring the time required for the block to rise 1°C. Typical values are 13 to 16 units, where each unit is equal to 200ms. A slow or long sensor lag can be due to a poor interface between thesensor and the block, such as lack of thermal grease, a poorly machinedsensor cavity or a faulty sensor.

The remaining install tests are currently executed by the installprogram but have limited diagnostic purposes due to the fact that theyare calculated values or are a function of so many variables that theirresults do not determine the source of a problem accurately.

The install program calculates the slope of the ramp cooling conductance(S_(c)) between 18° C. and 10° C. It is a measure of the linearity ofthe conductance curve. It is also used to approximate the ramp coolingconductance at 0° C. Typical values are 0.40±0.2. The spread in valuesattest to the fact that it is just an approximation.

    S.sub.c =(Kc.sub.-- 18°-Kc.sub.-- 10°)/(18° C.-10° C.)                                         (13)

The install program also calculates the cooling conductance K_(c0).K_(c0) is an approximation of the cooling conductance at 0° C. The valueis extrapolated from the actual conductance at 10° C. Typical values are23 watts/°C.±5. The formula used is:

    K.sub.c0 =Kc.sub.-- 10-(Sc*10° C.)

The install program also calculates coolant capacity (Cool Cp) which isan approximation of thermal capacity of the entire coolant stream(coolant, plumbing lines, heat exchanger, and valves). The coolingcapacity is equal to components that pump heat into the coolant minusthe components that remove heat from the coolant. The mechanics used tomeasure and calculate these components are complex and are described indetail in the source code description section. In this measurement, thecoolant is allowed to stabilize at 10° C. Maximum heater power isapplied to the sample block for a period of 128 seconds. ##STR1##

Characters enclosed in {} indicate the variable names used in the sourcecode.

Heater-Ping Test Pseudocode:

The heater ping test verifies that the heaters are properly wired forthe current line voltage.

Get the sample block and coolant to a known and stable point.

Turn ON the ramp cooling valves

Wait for the block and coolant to go below 5° C.

Turn OFF ramp cooling valves

Measure the cooling effect of control cooling by measuring the blocktemperature drop over a 10 second time interval. Wait 10 seconds forstabilization before taking any measurements.

Wait 10 seconds

temp1=block temperature

Wait 10 seconds

temp2=block temperature

{tempa}=temp2-temp1

Examine the variable {linevolts} which contains the actual measured linevoltage. Pulse the heater with 75 watts for a line voltage greater thanor equal to 190 V or with 300 watts if it less than or equal to 140 V.##STR2##

Measure the temperature rise over a 10 second time period. The result isthe average heat rate in 0.01°/second.

temp1=block temperature

Wait 10 seconds

temp2=block temperature {tempb}=temp2-temp1

Subtract the average heat rate {tempb} from the control cooling effectto calculate true heating rate

    heat.sub.-- rate={tempb}-{tempa}                           (17)

Evaluate the heat₋₋ rate. For 220 V-230 V, the heat rate should be lessthan 0.30°/second. For 100 V-120 V the heat rate should be greater than0.30°/second. ##STR3## KCC₁₃ Test Pseudocode:

This test measures the control cooling conductance also known as K_(cc).

K_(cc) is measured at a block temperature of 60° C.

Drive block to 60° C.

Maintain block temperature at 60° C. for 300 seconds

Integrate the power being applied to the sample block heaters over a 30second time period. Measure and integrate the power required to maintainthe block temperature with control cooling bias. ##STR4##

Compute the conductance by dividing the power sum by the temperaturesum. Note that the units are 10 mW/° C.

    K.sub.cc =({main.sub.-- pwr.sub.-- sum}+{aux.sub.-- pwr.sub.-- sum})/{dt.sub.-- sum}                                     (18)

BLOCK₋₋ CP Test Pseudocode:

This test measures the sample block thermal capacity.

Drive the block to 35° C.

Control block temperature at 35° C. for 5 seconds and record initialtemperature.

initial₋₋ temp=block temperature

Deliver maximum power to heaters for 20 seconds while summing thedifference in block to coolant temperature as well as heater power.##STR5##

Compute the joules in cooling power due to control cooling which occursduring ramp.

    cool.sub.-- joule=Control cooling conductance (K.sub.cc)* {dt.sub.-- sum}

Compute the total joules applied to the block from the main heater andcontrol cooling. Divide by temp change over the interval to computethermal capacity.

    Block CP=ramptime*(heater power-cool.sub.-- joule)/ delta.sub.-- temp (21)

where: ramptime=20 seconds

heater power=500 Watts

COOL₋₋ PWR₋₋ 10:

This test measures the chiller power at 10° C.

Control the coolant temperature at 10° C. and stabilize for 120 secs.##STR6##

At this point, the coolant has been at 10° C. for 120 seconds and hasstabilized. Integrate, over 32 seconds, the power being applied tomaintain a coolant temperature of 10° C. ##STR7##

Compute the number of joules of energy added to the coolant mass duringthe integration interval. "(coolant temp-cool₋₋ init)" is the change incoolant temp during the integration interval. 550 is the Cp of thecoolant in joules, thus the product is in joules. It represents theextra heat added to the coolant which made it drift from setpoint duringthe integration interval. This error is subtracted below from the totalheat applied before calculating the cooling power.

    cool.sub.-- init=(coolant temp-cool.sub.-- init)*550J      (22)

Add the main power sum to the aux heater sum to get joules dissipated in32 seconds. Divide by 32 to get the average joules/sec.

    {main.sub.-- pwr.sub.-- sum}=({main.sub.-- pwr.sub.-- sum}+{aux.sub.-- pwr.sub.-- sum}-cool.sub.-- init/32                       (23)

Compute the chiller power at 1° C. by summing all the chiller powercomponents.

    Power.sub.10° C. =main.sub.-- power.sub.-- sum+PUMP PWR +(K.sub.-- AMB*delta.sub.-- temp.sub.-- sum)                         (24)

where:

{main₋₋ pwr₋₋ sum}=summation of heater power over interval

PUMP PWR=12 Watts, pump that circulates coolant

delta₋₋ temp₋₋ sum=summation of amb-coolant over interval

K₋₋ AMB=20 Watts/K, thermal conductance from cooling to ambient.

KC ₁₀ Test Pseudocode:

This test measures the ramp cooling conductance at 10° C.

Control the coolant temperature at 10° C. ±0.5 and allow it to stabilizefor 10 seconds.

At this point, the coolant is at setpoint and is being controlled.Integrate, over a 30 second time interval, the power being applied tothe heaters to maintain the coolant at 10° C. Sum the difference betweenthe block and coolant temperatures. ##STR8##

Compute the energy in joules delivered to the block over the summationperiod. Units are in 0.1 watts.

    {main.sub.-- pwr.sub.-- sum}={main.sub.-- pwr.sub.-- sum}+{aux.sub.-- pwr.sub.-- sum}                                           (25)

Divide the power sum by block-coolant temperature sum to get rampcooling conductance in 100 mW/K.

    Kc.sub.-- 10 =(main.sub.-- pwr.sub.-- sum}/{dt.sub.-- sum} (26)

COOL₋₋ PWR₋₋ 18 Test Pseudocode:

This test measures the chiller power at 18° C .

Get the sample block and coolant to a known and stable point. Controlthe coolant temperature at 18° C. and stabilize for 120 secs. ##STR9##

At this point the coolant has been at 18° C. for 120 seconds and hasstabilized. Integrate, over 32 seconds, the power being applied tomaintain a coolant temperature of 18° C. ##STR10##

Compute the number of joules of energy added to the coolant mass duringthe integration interval. "(coolant temp-cool₋₋ init)" is the change incoolant temp during the integration interval. 550 is the Cp of thecoolant in joules, thus the product is in joules. It represents theextra heat added to the coolant which made it drift setpoint during theintegration interval. This error is subtracted below from the total heatapplied before calculating the cooling power.

    cool.sub.-- init=(coolant temp-cool.sub.-- init)*550J      (27)

Add main power sum to aux heater sum to get joules dissipated in 32seconds. Divide by 32 to get the average joules/sec.

    {main.sub.-- pwr.sub.-- sum}=({main.sub.-- pwr.sub.-- sum}+{aux.sub.-- pwr.sub.-- sum}-cool.sub.-- init)/32                      (28)

Compute the chiller power at 18° C. by summing all the chiller powercomponents.

    Power.sub.18° C. =main.sub.-- power.sub.-- sum+PUMP PWR+(K.sub.-- AMB* delta.sub.-- temp.sub.-- sum)                        (29)

where:

{main₋₋ pwr₋₋ sum}=summation of heater power over interval

PUMP PWR=12 Watts, pump that circulates coolant

delta₋₋ temp₋₋ sum=summation of amb-coolant over interval

K₋₋ AMB=20 Watts/K, Thermal conductance from cooling to ambient.

KC₋₋ 18 Test Pseudocode:

This test measures the ramp cooling conductance at 18° C.

Control the coolant temperature at 18° C.±0.5 and allow it to stabilizefor 10 seconds.

At this point, the coolant is at setpoint and being controlled.Integrate, over a 30 second time interval, the power being applied tothe heaters to maintain the coolant at 18° C. Sum the difference betweenthe block and coolant temperature. ##STR11##

Compute the energy in joules delivered to the block over the summationperiod. Units are in 0.1 watts.

    {main.sub.-- pwr.sub.-- sum}={main.sub.-- pwr.sub.-- sum}+{aux.sub.-- pwr.sub.-- sum}

Divide power sum by block-coolant temperature sum to get ramp coolingconductance in 100 mW/K.

    Kc.sub.-- 18 ={main.sub.-- pwr.sub.-- sum}/{dt.sub.-- sum} (31)

SENLAG Test Pseudocode:

This test measures the sample block sensor lag.

Drive the block to 35° C. Hold within ±0.2° C. for 20 seconds thenrecord temperature of block.

{tempa}=block temperature

Deliver 500 watts of power to sample block.

Apply 500 watts of power for the next 2 seconds and count the amount ofiterations through the loop for the block temperature to increase 1° C.Each loop iteration executes every 200 ms, therefore actual sensor lagis equal to count * 200 ms. ##STR12## Coolant CP Test Pseudocode:

This test computes the coolant capacity of the entire system.

Stabilize the coolant temperature at 10° C.±0.5.

Send message to the PID control task to ramp the coolant temperaturefrom its current value (about 10° C.) to 18° C.

Wait for the coolant to cross 12° C. so that the coolant CP ramp alwaysstarts at the same temperature and has clearly started ramping. Note theinitial ambient and block temperatures. ##STR13##

For the next two minutes, while the coolant temperature is ramping to18° C., sum the coolant temperature and the difference between theambient and coolant temperatures. ##STR14##

Calculate the change in temperatures over the two minute period.

    {blk.sub.-- delta}=block temperature-{blk.sub.-- delta}    (34)

    {h2o.sub.-- delta}=coolant temperature-{h2o.sub.-- delta}  (35)

Compute KChill, i.e., the rate of change of chiller power with coolanttemperature over the coolant range of 10° C. to 20° C. Note that unitsare in watts/10° C.

    Kchill=(Chiller Pwr @18° C.-Chiller Pwr @10° C.)(36)

Compute Sc which is the slope of the ramp cooling conductivity versusthe temperature range of 18° C. to 10° C. The units are in watts/10°C./10° C.

    Sc =(Kc.sub.-- 18-Kc.sub.-- 10)/8                          (37)

Compute Kc₋₋ 0, the ramp cooling conductance extrapolated to 0°C.

    Kc.sub.-- 0=Kc.sub.-- 10-(Sc*10)                           (38)

Compute Cp₋₋ Cool, the Cp of the coolant by: ##STR15##

where:

HEATPOWER=500 W, the heater power applied to warm the block, thusheating the coolant. It is multiplied by 128, as the heating intervalwas 128 secs.

PUMP_(PWR) =12 W, the power of the pump that circulates the coolantmultiplied by 128 seconds.

Pwr₋₋ 0° C.=The chiller power at 0° C. multiplied by 128 seconds.

Block₋₋ Cp=Thermal capacity of sample block.

blk₋₋ delta =Change in block temp over the heating interval.

K₋₋ AMB=20 Watts/K, thermal conductance from cooling to ambient.

temp₋₋ sum=The sum once per second of ambient-coolant temperature overthe interval.

h2o₋₋ delta=Change in coolant temperature over interval of heating(approximately 6° C.).

Kchill=Slope of chiller power versus coolant temp.

cool₋₋ sum=The sum of coolant temp, once per second, over the heatinginterval.

REAL TIME OPERATING SYSTEM-CRETIN

CRETIN is a stand alone, multitasking kernel that provides systemservices to other software modules called tasks. Tasks are written inthe "C" language with some time critical areas written in Intel 8085assembler. Each task has a priority level and provides an independentfunction. CRETIN resides in low memory and runs after the startupdiagnostics have successfully been executed.

CRETIN handles the task scheduling and allows only one task to run at atime. CRETIN receives all hardware interrupts thus enabling waitingtasks to run when the proper interrupt is received. CRETIN provides areal time clock to allow tasks to wait for timed events or pause forknown intervals. CRETIN also provides intertask communication through asystem of message nodes.

The firmware is composed of nine tasks which are briefly described inpriority order below. Subsequent sections will describe each task ingreater detail.

1. The control task (PID) is responsible for controlling the sampleblock temperature.

2. The keyboard task is responsible for processing keyboard input fromthe keypad.

3. The timer task waits for a half second hardware interrupt, then sendsa wake up message to both the sequence and the display task.

4. The sequence task executes the user programs.

5. The pause task handles programmed and keypad pauses when a program isrunning.

6. The display task updates the display in real time.

7. The printer task handles the RS-232 port communication and printing.

8. The LED task is responsible for driving the heating LED. It is alsoused to control the coolant temperature while executing Install.

9. The link task starts files that are linked together in a method bysimulating a keystroke.

Block Temperature Control Program (PID Task)

The Proportional Integral Differential (PID) task is responsible forcontrolling the absolute sample block temperature to 0.1° C., as well ascontrolling the sample block temperature non-uniformity (TNU, defined asthe temperature of the hottest well minus the temperature of the coldestwell) to less than ±0.5° C. by applying more heating power to theperimeter of the block to compensate for losses through the guard bandedges. The PID task is also responsible for controlling the temperatureof the heated cover to a less accurate degree. This task runs 5 timesper second and has the highest priority.

The amount of heating or cooling power delivered to the sample block isderived from the difference or "error" between the user specified sampletemperature stored in memory, called the setpoint, and the currentcalculated sample temperature. This scheme follows the standard loopcontrol practice. In addition to a power contribution to the filmheaters directly proportional to the current error, i.e., theproportional component, (setpoint temperature minus sample blocktemperature), the calculated power also incorporates an integral termthat serves to close out any static error (Setpoint temperature-Blocktemperature less than 0.5° C.). This component is called the integralcomponent. To avoid integral term accumulation or "wind-up",contributions to the integral are restricted to a small band around thesetpoint temperature. The proportional and integral component gains havebeen carefully selected and tested, as the time constants associatedwith the block sensor and sample tube severely restrict the system'sphase margin, thus creating a potential for loop instabilities. Theproportional term gain is P in Equation (46) below and the integral termgain is Ki in Equation (48) below.

The PID task uses a "controlled overshoot algorithm" where the blocktemperature often overshoots its final steady state value in order forthe sample temperature to arrive at its desired temperature as rapidlyas possible. The use of the overshoot algorithm causes the blocktemperature to overshoot in a controlled manner but does not cause thesample temperature to overshoot. This saves power and is believed to benew in PCR instrumentation.

The total power delivered to all heater of the sample block to achieve adesired ramp rate is given by:

    Power=(CP/ramp.sub.-- rate)+bias                           (40)

where:

CP=Thermal mass of block

bias=bias or control cooling power

ramp₋₋ rate=T_(final) -T_(initial) /desired ramp rate

This power is clamped to a maximum of 500 watts of heating power forsafety.

With every iteration of the task (every 200 ms) the system appliesheating or ramp cooling power (if necessary) based on the followingalgorithms.

The control system is driven by the calculated sample temperature. Thesample temperature is defined as the average temperature of the liquidin a thin walled plastic sample tube placed in one of the wells of thesample block (hereafter the "block"). The time constant of the system(sample tube and its contents) is a function of the tube type andvolume. At the start of a run, the user enters the tube type and theamount of reaction volume. The system computes a resultant time constant(τ or tau). For the MicroAmp™ tube and 100 microliters of reactionvolume, tau is approximately 9 seconds.

    T.sub.blk-new =T.sub.blk +Power*(200 ms/CP)                (41)

    T.sub.samp-new =T.sub.samp +(T.sub.blk-new -T.sub.samp)*200 ms/tau (42)

where:

T_(blk-new) =Current block temperature

T_(blk) =Block temperature 200 ms ago

Power=Power applied to block

CP=Thermal mass of block

T_(samp-new) =Current sample temperature

T_(samp) =Sample temperature 200 ms ago

tau=Thermal Time Constant of sample tube, adjusted for sensor lag(approximately 1.5)

The error signal or temperature is simply:

    error=Setpoint-T.sub.samp-new                              (43)

As in any closed loop system, a corrective action (heating or coolingpower) is applied to close out part of the current error. In Equation(45) below, F is the fraction of the error signal to be closed out inone sample period (200 mS).

    T.sub.samp-new T.sub.samp +F*(SP-T.sub.samp)               (44)

where SP=the user setpoint temperature

Due to the large lag in the system (long tube time constant), thefraction F is set low.

Combining formulas (42) and (44) yields:

    T.sub.samp-new =T.sub.samp +(T.sub.blk-new -T.sub.samp)*0.2/tau=T.sub.samp +F* (SP-T.sub.samp)                                       (45)

Combining formulas (41) and (45) and adding a term P (the proportionalterm gain) to limit block temperature oscillations and improve systemstability yields:

    Pwr=CP*P/T*((SP-T.sub.samp)*F*tau/T+T.sub.samp -T.sub.blk) (46)

where

p=the proportional term gain and

T=the sample period of 0.2 seconds (200 msec). and

P/T=1 in the preferred embodiment

Equation (46) is a theoretical equation which gives the power (Pwr)needed to move the block temperature to some desired value withoutaccounting for losses to the ambient through the guardbands, etc.

Once the power needed to drive the block is determined via Equation(46), this power is divided up into the power to be delivered to each ofthe three heater zones by the areas of these zones. Then the losses tothe manifolds are determined and a power term having a magnitudesufficient to compensate for these losses is added to the amount ofpower to be delivered to the manifold heater zone. Likewise, anotherpower term sufficient to compensate for power lost to the block supportpins, the block temperature sensor and the ambient is added to the powerto be delivered to the edge heater zones. These additional terms and thedivision of power by the area of the zones convert Equation (46) toEquations (3), (4) and (5) given above.

Equation (46) is the formula used by the preferred embodiment of thecontrol system to determine the required heating or cooling power to thesample block.

When the computed sample temperature is within the "integral band",i.e., ±0.5° C. around the target temperature (SP), the gain of theproportional term is too small to close out the remaining error.Therefore an integral term is added to the proportional term to closeout small errors. The integral term is disabled outside the integralband to prevent a large error signal from accumulating. The algorithminside the "integral band" is as follows:

    Int.sub.-- sum (new)=Int.sub.-- sum (old)+(SP-T.sub.samp)  (47)

    pwr.sub.-- adj=ki*Int.sub.-- sum (new)                     (48)

where,

Int₋₋ sum=the sum of the sample period of the difference between the SPand T_(SAMP) temperature, and

Ki=the integral gain (512) in the preferred embodiment).

Once a heating power has been calculated, the control softwaredistributes the power to the three film heater zones 254, 262 and 256 inFIG. 13 based on area in the preferred embodiment. The edge heatersreceive additional power based upon the difference between the blocktemperature and ambient temperature. Similarly, the manifold heatersreceive additional power based upon the difference between the blocktemperature and the coolant temperature.

Characters enclosed in {}in the pseudocode given below for the PID taskcorrespond to the variable names used in the source code of MicroficheAppendix C.

PID Pseudocode

Upon System Power up or Reset

Initialize PID variables

Read the line frequency

Initialize PIT and system clock

Turn off ramp cooling

Turn off all heaters

Calculate heater resistances

Do Forever-executes every 200 ms

If (block temperature>105) then

Turn off heaters

Turn on ramp valves

Display error message

Read the line voltage {linevolts}

Read the coolant sensor and convert to temperature {h2otemp}

Read the ambient sensor and convert to temperature {ambtemp}

Read the heated cover sensor and convert to temperature {cvrtemp}

Read the sample block sensor and convert to temperature {blktemp}.

This portion of the code also reads the temperature stable voltagereference and compares the voltage to a reference voltage that wasdetermined during calibration of the instrument. If there is anydiscrepancy, the electronics have drifted and the voltage readings fromthe temperature sensors are adjusted accordingly to obtain accuratetemperature readings. Compute the sample temperature {tubetenths} or thetemperature that gets displayed by using a low-pass digital filter.

    tubetenths=TT.sub.n-1 +(TB.sub.n -TT.sub.n-1)*T/tau        (49)

where TT_(n-1) =last sample temp {tubetenths}

TB_(n) =current block sensor temp {blktenths}

T=sample interval in seconds =200ms

tau=tau tube {cf₋₋ tau}-tau sensor {cf₋₋ lag}

Equation (49) represents the first terms of a Taylor series expansion ofthe exponential that defines the calculated sample temperature given asEquation (6) above.

Compute the temperature of the foam backing underneath the sample block,{phantenths} known as the phantom mass. The temperature of the phantommass is used to adjust the power delivered to the block to account forheat flow in and out of the phantom mass. The temperature is computed byusing a low pass digital filter implemented in software.

    phantenths=TT.sub.n-1 +(TB.sub.n -TT.sub.n-1)*T/tau        (50)

where TT_(n-1) =Last phantom mass temp {phantenths}

TB_(n) =Current block sensor temp {blktenths}

T=Sample interval in seconds =200 ms

tau_(foam) =Tau of foam block=30 secs.

Compute the sample temperature error (the difference between the sampletemperature and the setpoint temperature) {abs₋₋ tube₋₋ err}.

Determine ramp direction {fast₋₋ ramp}=UP₋₋ RAMP or DN₋₋ RAMP

If (sample temperature is within ERR of setpoint (SP)) then

PID not in fast transition mode. {fast₋₋ ramp}=OFF

where ERR=the temperature width of the "integral band", i.e., the errorband surrounding the target or setpoint temperature.

Calculate current control cooling power {cool₋₋ ctrl} to determine howmuch heat is being lost to the bias cooling channels.

Calculate current ramp cooling power {cool₋₋ ramp}

Calculate {cool₋₋ brkpt}. {cool₋₋ brkpt} is a cooling power that is usedto determine when to make a transition from ramp to control cooling ondownward ramps. It is a function of block and coolant temperature.

The control cooling power {cool₋₋ ctrl} and the ramp cooling power{cool₋₋ ramp} are all factors which the CPU must know to controldownward temperature ramps, i.e., to calculate how long to keep the rampcooling solenoid operated valves open. The control cooling power isequal to a constant plus the temperature of the coolant times thethermal conductance from the block to the bias cooling channels.Likewise, the ramp cooling power is equal to the difference between theblock temperature and the coolant temperature times the thermalconductance from the block to the ramp cooling channels. The coolingbreakpoint is equal to a constant (given in Microfiche Appendix C) timesthe difference in temperature between the block and the coolant.

Calculate a heating or cooling power {int₋₋ pwr} needed to move theblock temperature from its current temperature to the desired setpoint(SP) temperature.

    {int.sub.-- pwr}=KP*CP*[(SP-T.sub.SAMP)*{cf.sub.-- kd}+Ts-T.sub.BLK ](51)

where:

KP=Proportional gain=P/T in Equation (46)=approximately one in thepreferred embodiment

CP=Thermal mass of block

SP=Temperature setpoint

T_(SAMP) =Sample temperature

T_(BLK) =Block temperature

cf₋₋ kd=Tau*K_(d) /Delta₋₋ t where tau is the same tau as used inEquation (49) and K_(d) is a constant given in Microfiche Appendix C andDelta₋₋ t is the 200 msec sample period.

If (sample temperature is within {cf₋₋ iband} of setpoint) then##STR16##

Calculate the integral term power.

    integral term={i.sub.-- sum}*constant {cf.sub.-- term}.    (53)

Add the integral term to the power.

    {int.sub.-- pwr}={int.sub.-- pwr}+integral term            (54)

Adjust power to compensate for heating load due to the effects of thephantom mass (foam backing) by first finding the phantom mass power thenadding it to power {int₋₋ pwr}.

Calculate phantom mass power {phant₋₋ pwr} by:

    phant.sub.-- pwr=C*(blktenths-phantenths)/10               (55)

where: C=thermal mass of foam backing (1.0 W/K) Adjust heater power

    {int.sub.-- pwr}={int.sub.-- pwr}+{phant.sub.-- pwr}

Compute power needed in manifold heaters {auxl₋₋ power{ which willcompensate for loss from the sample block into the manifold edges thathave coolant flowing through it. Note that if the system is in adownward ramp, {aux1₋₋ power}=0.

The manifold zone power required is described below:

    {aux1.sub.-- power}=K1*(T.sub.BLK -T.sub.AMB)+K2*(T.sub.BLK -T.sub.COOL)+K5*(dT/dt)                                   (57)

where:

K1=Coefficient {cf₋₋ 1coeff}

K2=Coefficient {cf₋₋ 2coeff}

K5=Coefficient {cf₋₋ 5coeff}

dT/dt=Ramp rate

T_(BLK) =Block temperature

T_(AMB) =Ambient temperature

T_(COOL) =Coolant temperature

Compute power needed in edge heaters {aux2₋₋ power} which willcompensate for losses from the edges of the sample block to ambient.Note that if we are in a downward ramp {aux2₋₋ power}=0. The edge zonepower required is described below:

    {aux2.sub.-- power}=K3*(T.sub.BLK -T.sub.AMB)+K4*(T.sub.BLK -T.sub.COOL)+K6*(dT/dt)                                   (58)

where:

K3=Coefficient {cf₋₋ 3coeff}

K4=Coefficient {cf₋₋ 4coeff}

K6=Coefficient {cf₋₋ 6coeff}dT/dt=Ramp rate

T_(BLK) =Block temperature

T_(AMP) =Ambient temperature

T_(COOL) =Coolant temperature

Delete contribution of manifold {aux1₋₋ power} and edge heater power{aux2₋₋ power} to obtain total power that must be supplied by mainheaters and coolers.

    {int.sub.-- pwr}={int.sub.-- pwr}-{aux1.sub.- power}-{aux2.sub.-- power}(59)

Decide if the ramp cooling should be applied. Note that

{cool₋₋ brkpt} is used as a breakpoint from ramp cooling to controlcooling.

If (int₋₋ pwr<-cool₋₋ brkpt and performing downward ramp) to decidewhether block temperature is so much higher than the setpointtemperature that ramp cooling is needed then

Turn ON ramp valves else

Turn OFF ramp valves and depend upon bias cooling

At this point, {int₋₋ pwr} contains the total heater power and {aux1₋₋power} and {aux2₋₋ power} contain the loss from the block out to theedges. The power supplied to the auxiliary heaters is composed of twocomponents: aux₋₋ power and int₋₋ power. The power is distributed {int₋₋pwr} to the main and auxiliary heaters based on area.

total₋₋ pwr=int₋₋ pwr

int₋₋ pwr=total₋₋ pwr*66%

aux1₋₋ power=total₋₋ pwr*20%+aux1₋₋ power

aux2₋₋ power=total₋₋ pwr*14%+aux2₋₋ power

Compute the number of half cycles for the triac to conduct for each endzone and each iteration of the control loop to send the appropriateamount of power to the heaters. This loop executes once every 1/5second, therefore there are 120/5=24 half cycles at 60 Hz or 100/5=20 at50Hz. The number of half cycles is a function of requested power {int₋₋pwr}, the current line voltage {linevolts} and the heater resistance.Since the exact power needed may not be delivered each loop, a remainderis calculated {delta₋₋ power} to keep track of what to include from thelast loop.

    int.sub.-- pwr=int.sub.-- pwr+delta.sub.-- power           (60)

Calculate the number of 1/2 cycles to keep the triac on. Index is equalto the number of cycles to keep the triac on.

    index=power*main heater ohms*[20 or 24]/linevolts squared where Equation (61) is performed once for each heater zone and where "power"=int.sub.-- pwr for the main heater zone, aux1.sub.-- pwr for the manifold heater zone and aux2.sub.-- pwr for the edge heater zone.             (61)

Calculate the amount of actual power delivered.

    actual.sub.-- power=linevolts squared*index/main heater resistance (62)

Calculate the remainder to be added next time.

    delta.sub.-- power=int.sub.-- pwr-actual.sub.-- power      (63)

Calculate the number of 1/2 cycles for the edge and manifold heatersusing the same technique described for the main heaters by substituting{aux1₋₋ pwr} and {aux2₋₋ pwr} into Equation (60).

Load the calculated counts into the counters that control the main,manifold and edge triacs.

Look at heated cover sensor. If heated cover is less than 100° C., thenload heated cover counter to supply 50 watts of power.

Look at sample temperature If it is greater than 50° C., turn on HOT LEDto warn user not to touch block. END OF FOREVER LOOP

Keyboard Task

The purpose of the keyboard task is to wait for the user to press a keyon the keypad, compare the key to a list of valid keystrokes for thecurrent state, execute the command function associated with the validkey and change to a new state. Invalid keystrokes are indicated with abeep and then ignored. This task is the heart of the state driven userinterface. It is "state driven" because the action taken depends on thecurrent state of the user interface.

Keyboard Task Pseudocode:

Initialize keyboard task variables.

Turn off the cursor.

If (install flag not set) then Run the install program.

Send a message to pid task to turn on the heated cover.

If (the power failed while the user was running a program) then Computeand display the number of minutes the power was off for.

Write a power failure status record to the history file.

Send a message to the sequence task to start a 4° C. soak.

Give the user the option of reviewing the history file.

If (the user request to review the history file) then Go to the historyfile display.

Display the top level screen.

Do Forever

Send a message to the system that this task is waiting for a hardwareinterrupt from the keypad.

Go to sleep until this interrupt is received.

When awakened, read and decode the key from the keypad.

Get a list of the valid keys for the current state.

Compare the key to the list of valid keys.

If (the key is valid for this state) then Get the "action" and nextstate information for this key.

Execute the "action" (a command function) for this state.

Go to the next state.

Else

Beep the beeper for an invalid key.

End of Forever Loop

Timer Task Overview

The purpose of the timer task is to wake up the sequence and the realtime display task every half a second. The timer task asks the system(CRETIN) to wake it up whenever the half second hardware interrupt thatis generated by the clock/calendar device is received. The timer taskthen in turn sends 2 wake up messages to the sequence task and the realtime display task respectively. This intermediate task is necessarysince CRETIN will only service one task per interrupt and thus only thehigher priority task (the sequence task) would execute.

Timer Task Pseudocode:

Do Forever

Send a message to the system that this task is waiting for a hardwareinterrupt from the clock/calendar device.

Go to sleep until this interrupt is received.

When awakened, send a message to the sequence and to the real timedisplay task.

End Forever Loop

Sequence Task Overview

The purpose of the sequence task is to execute the contents of a userdefined program. It sequentially steps through each setpoint in a cycle,consisting of a ramp and a hold segment, and sends out setpointtemperature messages to the pid task which in turn controls thetemperature of the sample block. At the end of each segment, it sends amessage to the real time display task to switch the display and amessage to the printer task to print the segment's runtime information.The user can pause a running program by pressing the PAUSE key on thekeypad then resume the program by pressing the START key. The user canprematurely abort a program by pressing the STOP key. This task executesevery half a second when it is awakened by the timer task.

Sequence Task Pseudocode:

Do Forever

Initialize sequence task variables.

Wait for a message from the keyboard task that the user has pressed theSTART key or selected START from the menu or a message from link taskthat the next program in a method is ready to run. Go to sleep untilthis message is received. When awakened, update the ADC calibrationreadings to account for any drift in the analog circuitry.

If (not starting the 4° C. power failure soak sequence) then

Send a message to the printer task to print the PE title line, systemtime and date, program configuration parameters, the program type andits number.

If (starting a HOLD program) then

Get the temperature to hold at {hold₋₋ tp}.

Get the number of seconds to hold for {hold₋₋ time}.

If (ramping down more than 3° C. and {hold₋₋ tp}>45° C.) then Post anintermediate setpoint.

Else

Post the final setpoint {hold₋₋ tp}. While (counting down the hold time{hold₋₋ time{)

Wait for half second wake up message from timer task.

Check block sensor for open or short.

If (keyboard task detected a PAUSE key) then Post a setpoint of currentsample temp.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Post pre-pause setpoint.

If (an intermediate setpoint was posted) then Post the final setpoint.

If (the setpoint temp is below ambient temp and will be there for morethan 4 min.) then

Set a flag to tell pid task to turn off the heated cover.

Increment the half second hold time counter {store₋₋ time}.

Post the final setpoint again in case the hold time expired before theintermediate setpoint was reached-this insures the correct setpoint willbe written the history file. Write a data record to the history file.Send a message to the printer task to print the HOLD info. End of HOLDprogram

Else if (starting a CYCLE program) then

Add up the total number of seconds in a cycle {secs₋₋ in₋₋ run}, takinginto account the instrument ramp time and the user programmed ramp andhold times.

Get the total number of seconds in the program by multiplying the numberof seconds in a cycle by the number of cycles in a program {num₋₋ cyc}.

Total {secs₋₋ in₋₋ run}={secs₋₋ in₋₋ run}per cycle*{num₋₋ cyc}.

While (counting down the number of cycles {num₋₋ cyc})

While (counting down the number of setpoints {num₋₋ seg})

Get the ramp time {ramp₋₋ time}.

Get the final setpoint temp {t₋₋ final}.

Get the hold time {local₋₋ time}.

Send a message to the real time display task to display the ramp segmentinformation.

If (the user programmed a ramp time) then Compute the error {ramp₋₋ err}between the programmed ramp time and the actual ramp time as follows.This equation is based on empirical data.

    {ramp.sub.-- err}=prog ramp.sub.-- rate*15+0.5(up ramp)

    {ramp.sub.-- err}=prog ramp.sub.-- rate*6+1.0(down ramp)

where:

prog ramp₋₋ rate=(abs(T_(f) -T_(c))-1)/{ramp₋₋ time}

T_(f) =setpoint temp {t₋₋ final}

T_(c) =current block temp {blktemp}

abs=absolute value of the expression

Note: the '-1' is there because the clock starts within 1° C. ofsetpoint.

new ramp₋₋ time=old{ramp₋₋ time}-{ramp₋₋ err}

If (new ramp₋₋ time>old {ramp₋₋ time}) then new ramp₋₋ time=old {ramp₋₋time}.

Else new ramp₋₋ time=0.

While (sample temp is not within a user configured temp {cf₋₋ clk₋₋dev}of setpoint) Wait for half second wake up message from timer task.Post a new ramp setpoint every second.

Else if (ramping down more than 3° C. and {t₋₋ final}>45° C.) then

Post an intermediate setpoint.

While (sample temp is not within a user configured temp {cf₋₋ clk₋₋ dev}of setpoint) Wait for half second wake up message from, timer task.Increment the half second ramp time counter. Check block sensor for openor short. If (keyboard task detected a PAUSE key) then

Post a setpoint of current sample temp.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Post pre-pause setpoint.

Post the final setpoint.

While (sample temp is not within a user configured temp {cf₋₋ clk₋₋ dev}of setpoint) Wait for half second wake up message from timer task.

Increment the half second ramp time counter.

Check block sensor for open or short.

If (keyboard task detected a PAUSE key) then Post a setpoint of currentsample temp.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Post pre-pause setpoint.

Send a message to the printer task to print the ramp information.

Beep beeper to signal end of ramp segment.

Send a message to the real time display task to display the hold segmentinformation.

While (counting down the hold time)

Wait for half second wake up message from timer task.

Increment the half second hold time counter.

Check block sensor for open or short.

If (keyboard task detected a PAUSE key) then

Post a setpoint of current sample temp.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Post pre-pause setpoint.

Write a data record to the history file.

Send a message to the printer task to print the hold information.

If (the final setpoint temp has drifted more than the user configurableamount {cf₋₋ temp₋₋ dev}) then

Write an error record to the history file.

Check for a programmed pause and execute if necessary.

Go to next segment.

Send a message to the printer task to print an end of cycle message.

Go to next cycle. End of CYCLE program.

Else if (starting an AUTO-CYCLE program) then Add up the total number ofseconds in each program {secs₋₋ in₋₋ run} taking into account theinstrument ramp time and the user programmed hold times and temperatureswhich can be automatically incremented or decremented by a programmedamount each cycle. While (counting down the number of cycles {num₋₋cyc})

While (counting down the number of setpoints {num₋₋ seg}) Get the finalsetpoint temp {t₋₋ final}. Get the hold time{time₋₋ hold}.

Check if the user programmed an auto increment or decrement of thesetpoint temp and/or the hold time and adjust them accordingly. If (theauto increment or decrement of the temp causes the setpoint to go below0° C. or above 99.9° C.) then An error record is written to the historyfile.

The setpoint is capped at either 0° C. or 99.9° C. If (the autodecrement of the hold time causes the hold time to go below 0 seconds)then An error record is written to the history file.

The hold time is capped at 0° C. Send a message to real time displaytask to display the ramp segment information. If (ramping down more than3° C. and {t₋₋ final}>45° C.) then Post an intermediate setpoint.

While (sample temp is not within a user configured temp {cf₋₋ clk₋₋ dev}of setpoint) Wait for half second wake up message from timer task.Increment the half second ramp time counter. Check block sensor for openor short. If (keyboard task detected a PAUSE key) then Post a setpointof current sample temp. Send a message to wake up the pause task. Go tosleep until awakened by the pause task. Post pre-pause setpoint. Postthe final setpoint.

While (sample temp is not within a user configured temp {cf₋₋ clk₋₋ dev}of setpoint) Wait for half second wake up message from timer task.Increment the half second ramp time counter. Check block sensor for openor short.

If (keyboard task detected a PAUSE key) then Post a setpoint of currentsample temp. Send a message to wake up the pause task. Go to sleep untilawakened by the pause task. Post pre-pause setpoint.

Send a message to the printer task to print the ramp segmentinformation. Beep beeper to signal end of ramp portion of segment. Senda message to the real time display task to display the hold segmentinformation. While (counting down the hold time) Wait for half secondwake up message from timer task.

Increment the half second hold time counter. Check block sensor for openor short. If (keyboard task detected a PAUSE key) then Post a setpointof current sample temp. Send a message to wake up the pause task. Go tosleep until awakened by the pause task. Post pre-pause setpoint.

Write a data record to the history file. Send a message to the printertask to print the hold information. If (the final setpoint temp hasdrifted more than the user configurable amount {cf₋₋ temp₋₋ dev}) then

Write an error recored to the history file.

Go to next segment.

Send a message to the printer task to print an end of cycle message.

Go to next cycle.

End of AUTO-CYCLE program.

Else if (starting a POWER FAILURE sequence) then

Post a setpoint of 4° C.

Set a flag {subamb₋₋ hold} so that the pid task will shut off the heatedcover.

DO FOREVER

Wait for a half second wake up message from the timer task.

Increment the half second hold time counter.

END FOREVER LOOP

End of power failure sequence

Write a run end status record to the history file.

If (running a method)

Set a flag {weird₋₋ flag} so the link task will know to send a messageto the sequence task to start the next program running.

Else

Return user interface to idle state display.

End of Forever Loop

Pause Task Overview

The purpose of the pause task is to handle either a pause that the userprograms in a CYCLE program or a pause when the user presses the PAUSEkey on the keypad.

When the sequence task encounters a programmed pause while executing aCYCLE program, it goes to sleep and awakens the pause task. The pausetask in turn sends a message to the real time display task tocontinually display and decrement the time the user asked to pause for.When the pause timer times out, the pause task sends a message to awakenthe sequence task and then goes to sleep. The user can prematurelyresume the program by pressing the START key on the keypad cr canprematurely abort the program by pressing the STOP key.

When the keyboard task detects a PAUSE key while a program is running,it sets a flag {pause₋₋ flag} then waits for the sequence task toacknowledge it. When the sequence task sees this flag set, it sends anacknowledgment message back to the keyboard task then puts itself tosleep. When the keyboard task receives this message, it awakens thepause task. The pause task sends a message to the real time display taskto continually display and increment the amount of time the program ispaused for. The timer will time out when it reaches the pause time limitset by the user in the configuration section. The user can resume theprogram by pressing the START key on the keypad or abort the program bypressing the STOP key.

Pause Task Pseudocode:

Do Forever

Wait for a message from the keyboard task indicating a keypad pause, ora message form the sequence task indicating a user programmed pause.

Go to sleep until a message is received.

When awakened, check a flag for the type of pause initiated.

If (it is a programmed pause) then

Send a message to the real time display task to display the pause timercounting up.

Else

Send a message to the real time display task to display the pause timercounting down.

While (counting down the time out counter)

Send a message to the system to suspend this task for half a second.

Send a message to the printer task to print the pause information.

If (it is a programmed pause) then

Write a status record to the history file.

The pause has timed out so send a message to the wake up the sequencetask.

Send a message to the real time display task to halt the pause display.

Send a message to the real time display task to resume the runningprogram display.

Else (it is a keypad pause)

The pause has timed out and the program must be aborted so send amessage to the system to halt the sequence task and send it back to thetop of its FOREVER loop.

If (the program running was a HOLD program)

Send a message to the printer task to print the hold information.

Write a status record to the history file.

Return the user interface to its idle state.

Display an abort message.

End of Forever Loop

Display Task Overview

The purpose of the real time display task is to display temperatures,timers, sensor readings, ADC channel readings, and other parameters thatneed to be continually updated every half second.

Display Task Pseudocode:

Initialize display task variables.

Do Forever

Wait for a message every half second from the timer task.

Go to sleep until the message is received.

When awakened, check if another task has sent a list of parameters todisplay or a flag to halt the current update.

Toggle the half second flag {half₋₋ sec}.

If (there's a list of parameters to display) then

Set a semaphore so no one else will update the display.

Turn off the cursor.

While (stepping through the list of parameters)

If (it is a time parameter) then

Display the time.

If (half second flag {half₋₋ sec} is set) then

Increment or decrement the time variable.

Else if (it is a decimal number) then

Display a decimal number.

Else if (it is an integer number) then

Display the integer.

Else if (it is an ADC channel readout) then

Read the counts from the ADC channel.

If (need it displayed as mV) then

Convert counts to mV.

Display the value.

Else if (it is a power display) then

Display the power in terms of watts.

Else if (it is the hours left parameter) then

Convert seconds to tenths of hours.

Display the hours left in tenths of hours.

If (half second flag {half₋₋ sec} is set) then

Decrement the seconds variable.

If (the cursor was on) then

Turn it back on.

Store the current system time in battery RAM.

Clear the semaphore to release the display.

End of Forever Loop

Printer Task Overview

The purpose of the printer task is to handle the runtime printing. It isa low priority task and should not interfere with other time criticaltasks.

Printer Task Pseudocode:

Do Forever

Wait for a message from another task that wishes to print.

Go to sleep until a message is received.

When awaken, make local copies of the global variables to be printed.

Post a printer acknowledgement message.

If (need to print a status or error message) then

Print the information contained in the current history record.

Else if (need to print the page header) then

Print the company name, instrument ID, firmware version number and thecurrent system time and date.

Else if (need to print the program header) then

Print the type of program and its number.

Else if (need to print the program configuration parameters) then

Print the tube type, reaction volume and the sample temperaturedeviation from setpoint that starts the clock.

Else if (need to print end of cycle information) then

Print the ending time and temperature.

Else if (need to print segment information) then

Print either the ramp or hold segment information.

Else if (need to print a pause status message) then

Print the amount of time paused for and at what temp.

End of Forever Loop

LED Task Overview

The purpose of the LED task is to make the illumination of the "Heating"LED reflect the power applied to the main heater. This is a low prioritytask that runs once a second.

LED Task Pseudocode:

Initialize LED task variables.

Do Forever

Send a message to the system to wake this task every second.

Go to sleep.

When awaken, load counter 2 of PIC timer A with a value that reflectsthe power applied to the main heater as follows:

load counter with value={K₋₋ htled}*{ht₋₋ led}

Where:

{K₋₋ htled} holds a constant to compute the time to pulse the heatingLED and is equal to 15200/500. 15200 is a little greater than the PIC'sclock of 14.4 KHz and this is the value loaded into the timer to keepthe LED constantly on. 500 is the main heater power.

{ht₋₋ led} will be a value between 0 and 500 and will be equal to thewatts applied to the main heater.

End of Forever Loop

Link Task Overview

The purpose of the link task is to simulate the user pressing the STARTkey on the keypad. This task is necessary so that programs can beexecuted one right after the other (as in a method) without userintervention. The link task wakes up the sequence task and it beginsrunning the next program as if the START key were pressed.

Link Task Pseudocode:

Initialize link task variables.

Do Forever

If (the flag {weird₋₋ flag} is set and it is not the first file in themethod) then

Send a message to the sequence task to wake up and run a program.

End of Forever Loop

Start Up Sequence POWER-UP SEQUENCE

When the power to the instrument is turned on or the software does aRESET, the following sequence takes place. Note: the numbers belowcorrespond to numbers on the flow chart in FIGS. 53 and 54.

1. Transmit a Ctrl-G (decimal 7) character out the RS-232 printer port.Poll the RS-232 port for at least 1 second and if a Ctrl-G is received,it is assumed that an external computer is attached to the port and allcommunication during the power-up sequence will be redirected from thekeypad to the RS-232 port. If no Ctrl-G is received, the power-upsequence continues as normal.

2. Check if the MORE key is depressed. If so, go straight to theservice-only hardware diagnostics.

3. The next 3 tests are an audio/visual check and cannot report anerror: 1) the beeper beeps 2) the hot, cooling, and heating LEDs on thekeypad are flashed 3) each pixel of the display is highlighted. Thecopyright and instrument ID screens are displayed as the power-updiagnostics execute.

4. Should an error occur in one of the power-up diagnostics, the name ofthe component that failed is displayed and the keypad is locked exceptfor the code `MORE 999` which will gain access to the service-onlyhardware diagnostics.

5. Check channel 0 of the PPI-B device to see if the automated test bitis pulled low. If it is, run the UART test. If the test passes, beep thebeeper continuously.

6. Start the CRETIN operating system which in turn will start up eachtask by priority level.

7. Check a flag in battery RAM to see if the instrument has beencalibrated. If not, display an error message and lock the keypad exceptfor the code `MORE 999` which will gain access to the service-onlycalibration tests.

8. Run a test that measures the voltage and line frequency and see ifboth these values match the configuration plug selected whilecalibrating the instrument. If not, display an error message and lockthe keypad except for the code `MORE 999` which will gain access to theservice-only calibration tests.

9. Perform the heater ping test as described in the Install section. Ifthe heaters are wired wrong, display an error message and lock thekeypad except for the code `MORE 999` which will gain access to theservice-only calibration tests.

10. Check a flag in battery RAM to see if the instrument has beeninstalled. If not, display an error message and lock the keypad exceptfor the code `MORE 999` which will gain access to the install routine.

11. If not in remote mode, check a flag in battery RAM to see if therewas a power failure while the instrument was running. If so, start a 4°C. soak and display the amount of time the power was off for. Ask theuser if they wish to view the history file which will tell them exactlyhow far along they were in the run when the power went off. If theyselect yes, they go straight to the user diagnostics.

12. Beep the beeper and clear the remote mode flag so all communicationnow is back through the keypad.

13. Check a flag in battery RAM to see if manufacturing wants their testprogram automatically started. If so, start the program running andreset the instrument after its done.

14. Display the top level user interface screen.

Electronics and Software Version 2

Referring to FIGS. 47A and 47B (hereafter FIG. 47), there is shown ablock diagram for the electronics of a preferred embodiment of a controlsystem in a class of control systems represented by CPU block 10 inFIG. 1. The purpose of the control electronics of FIG. 47 is, interalia, to receive and store user input data defining the desired PCRprotocol, read the various temperature sensors, calculate the sampletemperature, compare the calculated sample temperature to the desiredtemperature as defined by the user defined PCR protocol, monitor thepower line voltage and control the film heater zones and the rampcooling valves to carry out the desired temperature profile of the userdefined PCR protocol.

A microprocessor (hereafter CPU) 450 executes the control programdescribed below and given in Microfiche Appendix F in source code form.In the preferred embodiment, the CPU 450 is an OKI CMOS 8085. The CPUdrives an address bus 452 by which various ones of the other circuitelements in FIG. 47 are addressed. The CPU also drives a data bus 454 bywhich data is transmitted to various of the other circuit elements inFIG. 47.

The control program of Microfiche Appendix F and some system constantsare stored in EPROM 456. User entered data and other system constantsand characteristics measured during the install process (install programexecution described below) are stored in battery backed up RAM 458. Asystem clock/calendar 460 supplies the CPU 450 with date and timeinformation for purposes of recording a history of events during PCRruns and the duration of power failures as described below in thedescription of the control software.

An address decoder 462 receives and decodes addresses from the addressbus 452 and activates the appropriate chip select lines on a chip selectbus 464.

The user enters PCR protocol data via a keyboard 466 in response toinformation displayed by CPU on display 468. The two way communicationbetween the user and the CPU 450 is described in more detail below inthe user interface section of the description of the control software. Akeyboard interface circuit 470 converts user keystrokes to data which isread by the CPU via the data bus 454.

Two programmable interval timers 472 and 474 each contain counters whichare loaded with counts calculated by the CPU 450 to control theintervals during which power is applied to the various film heaterzones.

An interrupt controller 476 sends interrupt requests to the CPU 450every 200 milliseconds causing the CPU 450 to run the PID task describedbelow in the description of the control software. This task reads thetemperature sensors and calculates the heating or cooling powernecessary to move the sample temperature from its current level to thelevel desired by the user for that point in time in the PCR protocolbeing executed.

A UART 478 services an RS232 interface circuit 480 such that data storedin the RAM 480 may be output to a printer. The control softwaremaintains a record of each PCR run which is performed with respect tothe actual temperatures which existed at various times during the runfor purposes of user validation that the PCR protocol actually executedcorresponded to the PCR protocol desired by the user. In addition, userentered data defining the specific times and temperatures desired duringa particular PCR protocol is also stored. All this data and other dataas well may be read by the CPU 450 and output to a printer coupled tothe RS232 port via the UART 478. The RS232 interface also allows anexternal computer to simulate the keypad and display.

A programmable peripheral interface (hereafter PPI) 482 serves as aprogrammable set of 3 input/output registers. At power-up, the CPU 450selects the PPI 482 via the address decoder 462 and the chip select bus464. The CPU then writes a data word to the PPI via data bus 454 toprogram the PPI 482 regarding which registers are to be output ports andwhich are to be input ports. Subsequently, the CPU 450 uses the outputregisters to store data words written therein by the CPU via the databus 454 to control the internal logic state of a programmable arraylogic chip (PAL) 484.

The PAL 484 is a state machine which has a plurality of input signalsand a plurality of output signals. PAL's in general contain an array oflogic which has a number of different states. Each state is defined bythe array or vector of logic states at the inputs and each state resultsin a different array or vector of logic states on the outputs. The CPU450, PPI 482, PAL 484 and several other circuits to be defined belowcooperate to generate different states of the various output signalsfrom the PAL 484. These different states and associated output signalsare what control the operation of the electronics shown in FIG. 47 aswill be described below.

A 12 bit analog-to-digital converter (A/D) 486 converts analog voltageson lines 488 and 490 to digital signals on data bus 454. These are readby the CPU by generating an address for the A/D converter such that achip select signal on bus 464 coupled to the chip select input of theA/D converter goes active and activates the converter. The analogsignals on lines 488 and 490 are the output lines of two multiplexers492 and 494. Multiplexer 492 has four inputs ports, each having twosignal lines. Each of these ports is coupled to one of the fourtemperature sensors in the system. The first port is coupled to thesample block temperature sensor. The second and third ports are coupledto the coolant and ambient temperature sensors, respectively and thefourth port is coupled to the heated cover temperature sensor. A typicalcircuit for each one of these temperature sensors is shown in FIG. 48. A20,000 ohm resistor 496 receives at a node 497 a regulated +15 voltregulated power supply 498 in FIG. 47 via a bus connection line which isnot shown. This + 15 volts D.C. signal reverse biases a zener diode 500.The reverse bias current and the voltage drop across the zener diode arefunctions of the temperature. The voltage drop across the diode is inputto the multiplexer 292 via lines 502 and 504. Each temperature sensorhas a similar connection to the multiplexer 292.

Multiplexer 494 also has 4 input ports but only three are connected. Thefirst input port is coupled to a calibration voltage generator 506. Thisvoltage generator outputs two precisely controlled voltage levels to themultiplexer inputs and is very thermally stable. That is, the referencevoltage output by voltage source 506 drifts very little if at all withtemperature. This voltage is read from time to time by the CPU 450 andcompared to a stored constant which represents the level this referencevoltage had at a known temperature as measured during execution of theinstall process described below. If the reference voltage has driftedfrom the level measured and stored during the install process, the CPU450 knows that the other electronic circuitry used for sensing thevarious temperatures and line voltages has also drifted and adjuststheir outputs accordingly to maintain very accurate control over thetemperature measuring process.

The other input to the multiplexer 494 is coupled via line 510 to anRMS-to-DC converter circuit 512. This circuit has an input 514 coupledto a step-down transformer 516 and receives an A.C. voltage at input 514which is proportional to the then existing line voltage at A.C. powerinput 518. The RMS-to-DC converter 512 rectifies the A.C. voltage andaverages it to develop a D.C. voltage on line 510 which also isproportional to the A.C. input voltage on line 518.

Four optically coupled triac drivers 530, 532, 534 and 536 receive inputcontrol signals via control bus 538 from PAL logic 484. Each of thetriac drivers 530, 532 and 534 controls power to one of the three filmheater zones. These heater zones are represented by blocks 254, 260/262and 256/258 (the same reference numerals used in FIG. 13). The triacdriver 536 controls power to the heated cover, represented by block 544via a thermal cut-out switch 546. The heater zones of the film heaterare protected by a block thermal cutout switch 548. The purpose of thethermal cutout switches is to prevent meltdown of the film heater/sampleblock on the heated cover in case of a failure leading to the triacdrivers being left on for an unsafe interval. If such an event happens,the thermal cut-out switches detect an overly hot condition, and shutdown the triacs via signals on lines 552 or 554.

The main heater zone of the film heater is rated at 360 watts while themanifold and edge heater zones are rated at 180 watts and 170 wattsrespectively. The triac drivers are Motorola MAC 15A10 15 amp triacs.Each heater zone is split into 2 electrically isolated sections eachdissipating 1/2 the power. The 2 halves are connected in parallel forline voltages at 518 less than 150 volts RMS. For line voltages greaterthan this, the two halves are connected in series. These alternateconnections are accomplished through a "personality" plug 550.

The AC power supply for the film heater zones is line 559, and the ACsupply for the heated cover is via line 560.

A zero crossing detector 566 provides basic system timing by emitting apulse on line 568 at each zero crossing of the AC power on line 518. Thezero crossing detector is a National LM 311N referenced to analog groundand has 25 mV of hysteresis. The zero crossing detector takes its inputfrom transformer 516 which outputs A.C. signal from 0 to 5.52 volts foran A.C. input signal of from 0 to 240 volts A.C.

A power transformer 570 supplies A.C. power to the pump 41 that pumpscoolant through the ramp and bias cooling channels. The refrigerationunit 40 also receives its A.C. power from the transformer 570 viaanother portion of the personality plug 550. The transformer 550 alsosupplies power to three regulated power supplies 572, 498 and 574 andone unregulated power supply 576.

For accuracy purposes in measuring the temperatures, the calibrationvoltage generator 506 uses a series of very precise, thin-film, ultralowtemperature drift 20K ohm resistors (not shown in FIG. 47 but shown asresistors RA1 in the schematics of Microfiche Appendix E). These sameultralow drift resistors are used to set the gain of an analog amplifier578 which amplifies the output voltage from the selected temperaturesensor prior to conversion to a digital value. These resistors driftonly 5 ppm/C°.

All the temperature sensors are calibrated by placing them (separatedfrom the structures whose temperatures they measure) first in a stable,stirred-oil, temperature controlled bath at 40° C. and measuring theactual output voltages at the inputs to the multiplexer 492. Thetemperature sensors are then placed in a bath at a temperature of 95° C.and their output voltages are again measured at the same points. Theoutput voltage of the calibration voltage generator 506 is also measuredat the input of the multiplexer 494. For each temperature, the digitaloutput difference from the A/D converter 486 between each of thetemperature sensor outputs and the digital output that results from thevoltage generated by the calibration voltage generator 506 is measured.The calibration constants for each temperature sensor to calibrate eachfor changes in temperature may then be calculated.

The sample block temperature sensor is then subjected to a furthercalibration procedure. This procedure involves driving the sample blockto two different temperatures. At each temperature level, the actualtemperature of the block in 16 different sample wells is measured using16 RTD thermocouple probes accurate to within 0.02° C. An averageprofile for the temperature of the block is then generated and theoutput of the A/D converter 464 is measured with the block temperaturesensor in its place in the sample block. This is done at bothtemperature levels. From the actual block temperature as measured by theRTD probes and the A/D output for the block temperature sensor, afurther calibration factor can be calculated. The temperaturecalibration factors so generated are stored in battery backed up RAM458. Once these calibration factors are determined for the system, it isimportant that the system not drift appreciably from the electricalcharacteristics that existed at the time of calibration. It is importanttherefore that low drift circuits be selected and that ultralow driftresistors be used. The selections made for the analog components for anexemplary embodiment are given in Microfiche Appendix E.

The manner in which the CPU 450 controls the sample block temperaturecan be best understood by reference to the section below describing thecontrol program. However, to illustrate how the electronic circuitry ofFIG. 47 cooperates with the control software to carry out a PCR protocolconsider the following.

The zero crossing detector 566 has two outputs in output bus 568. One ofthese outputs emits a negative going pulse for every positive goingtransition of the A.C. signal across the zero voltage reference. Theother emits a negative pulse upon every negative-going transition of theA.C. signal across the zero reference voltage level. These two pulses,shown typically at 580 define one complete cycle or two half cycles. Itis the pulse trains on bus 568 which define the 200 millisecond sampleperiods. For 60 cycle/sec A.C. as found in the U.S., 200 millisecondscontains 24 half cycles.

A typical sample period is shown in FIG. 49. Each "tick" mark in FIG. 49represents one half cycle. During each 200 msec sample period, the CPU450 is calculating the amount of heating or cooling power needed tomaintain the sample block temperature at a user defined setpoint orincubation temperature or to move the block temperature to a newtemperature depending upon where in the PCR protocol time line theparticular sample period lies. The amount of power needed in each filmheater zone is converted into a number of half cycles each heater zoneis to remain off during the next 200 msec sample period. Just before theend of the current sample period in which these calculations are made,the CPU 450 addresses each of the 4 timers in the programmable intervaltimer (PIT) 472. To each timer, the CPU writes data constituting a"present" count representing the number of hafl cycles the heater zoneassociated with that timer is to remain off in the next sample period.In FIG. 49, this data is written to the timers during interval 590 justpreceding the starting time 592 of teh next sample period. Assume that arapid ramp up to the denaturation temperature of 94° C. is called for bythe user setppoint data for an interval which includes the sampleinterval between times 592 and 594. Accordingly, the film heaters willbe on for most of the period. Assume that the central zone heater is tobe on for all but three of the half cycles during the sample period. Inthis case, the CPU 450 writes a three into the counter in PIT 472associated with the central zone heater during interval 590. This writeoperation automatically causes the timer to issue a "shut off" signal onthe particular control line of bus 592 which controls the central zoneheater. This "shut off" signal couaes the PAL 484 to issue a "shut off"signal on the particular one of the signal lines in bus 538 associatedwith the central zone. The triac driver 530 then shuts off at the nextzero crossing, i.e., at time 592. The PIT receives a pulse train ofpositive-going pulses on line 594 from the PAL 484. These pulses aretranslations of the zero-crossing pulses on 2-line bus 568 by PAL 484into positive going pulses at all zero crossing pulses on 2-line bus 568by PAL 484 into positive going pulses at all zero crossings on a singleline, i.e., line 594. The timer in PIT 472 associated with the centralfilm heater zone starts counting down from its present count of 3 usingthe half cycle marking pulses on line 594 as its clock. At the end ofthe third half cycle, this timer reaches 0 and causes its output signalline on bus 592 to change states. This transition from the off to onestate is shown at 596 in FIG. 49. This transition is communicated to PAL484 and causes it to change the state of the appropriate output signalon bus 538 to switch the triac driver 530 on at the third zero-crossing.Note that by switching the triacs on at the zero crossings as is done inthe preferred embodiment, switching off of a high current flowingthrough an inductor (the film heater conductor) is avoided. Thisminimizes the generation of radio frequency interference or other noise.Note that the technique of switching a portion of each half cycle to thefilm heater in accordance with the calculated amount of power neededwill also work as an alternative embodiment, but is not preferredbecause of the noise generated by this technique.

The other timers of PIT 472 and 474 work in a similar manner to managethe power applied to the other heater zones and to the heated cover inaccordance with power calculated by the CPU.

Ramp cooling is controlled by CPU 450 directly through the peripheralinterface 482. When the heating/cooling power calculations performedduring each sample period indicate that ramp cooling power is needed,the CPU 450 addresses the programmable peripheral interface (PPI) 482. Adata word is then written into the appropriate register to drive outputline 600 high. This output line triggers a pair of monostablemultivibrators 602 and 604 and causes each to emit a single pulse, onlines 606 and 608, respectively. These pulses each have peak currentsjust under 1 ampere and a pulse duration of approximately 100milliseconds. The purpose of these pulses is to drive the solenoid valvecoils that control flow through the ramp cooling channels very hard toturn on ramp cooling flow quickly. The pulse on line 606 causes a driver610 to ground a line 612 coupled to one side of the solenoid coil 614 ofone of the solenoid operated valves. The other terminal of the coil 614is coupled to a power supply "rail" 616 at +24 volts DC from powersupply 576. The one shot 602 controls the ramp cooling solenoid operatedvalve for flow in one direction, and the one shot 604 controls thesolenoid operated valve for flow in the opposite direction.

Simultaneously, the activation of the RCOOL signal on line 600 causes adriver 618 to be activated. This driver grounds the line 612 through acurrent limiting resistor 620. The value of this current limitingresistor is such that the current flowing through line 622 is at leastequal to the hold current necessary to keep the solenoid valve 614 open.Solenoid coils have transient characteristics that require largecurrents to turn on a solenoid operated valve but substantially lesscurrent to keep the valve open. When the 100 msec pulse on line 606subsides, the driver 612 ceases directly grounding the line 612 leavingonly the ground connection through the resistor 620 and driver 618 forholding current.

The solenoid valve 614 controls the flow of ramp cooling coolant throughthe sample block in only 1/2 the ramp cooling tubes, i.e., the tubescarrying the coolant in one direction through the sample block. Anothersolenoid operated valve 624 controls the coolant flow of coolant throughthe sample block in the opposite direction. This valve 624 is driven inexactly the same way as solenoid operated valve 614 by drivers 626 and628, one shot 604 and line 608.

The need for ramp cooling is evaluated once every sample period. Whenthe PID task of the control software determines from measuring the blocktemperature and comparing it to the desired block temperature that rampcooling is no longer needed, the RCOOL signal on line 600 isdeactivated. This is done by the CPU 450 by addressing the PIC 482 andwriting data to it which reverses the state of the appropriate bit inthe register in PIC 482 which is coupled to line 600.

The logic equations for PAL 484 are attached hereto as MicroficheAppendix D. The logic equations for the address decoder 462, which isalso programmable array logic, are also attached hereto is MicroficheAppendix D.

The PIT 474 also has two other timers therein which time a 20 Hzinterrupt and a heating LED which gives a visible indication when thesample block is hot and unsafe to touch.

The system also includes a beeper one shot 630 and a beeper 632 to warnthe user when an incorrect keystroke has been made.

The programmable interrupt controller 476 is used to detect 6interrupts; Level 2-20 Hz; Level 3 - Transmit Ready; Level 4 - Receiveready; Level 5 - Keyboard interrupt; Level 6 - 2 Hz signal for thedisplay and sequence task; and, Level 7 - A.C. line zero cross.

The programmable peripheral interface 482 has four outputs (not shown)for controlling the multiplexers 492 and 494. These signals MUX1 EN andMUX2 EN enable one or the other of the two multiplexers 492 and 494while the signals MUX 0 and MUX 1 control which channel is selected forinput to the amplifier 578. These signals are managed so that only onechannel from the two multiplexers can be selected at any one time.

An RLTRIG* signal resets a timeout one shot 632 for the heaters whichdisables the heaters via activation of the signal TIMEOUT EN* to the PAL484 if the CPU crashes. That is, the one shot 632 has a predeterminedinterval which it will wait after each reset before it activates thesignal TIMEOUT EN* which disables all the heater zones. The CPU 450executes a routine periodically which addresses the PIC 482 and writesdata to the appropriate register to cause activation of a signal on line634 to reset the one shot 632. If the CPU 450 "crashes" for any reasonand does not execute this routine, the timeout one-shot 632 disables allthe heater zones.

The PIC 482 also has outputs COVHTR EN* and BLKHTREN* (not shown) forenabling the heated cover and the sample block heater. Both of thesesignals are active low and are controlled by the CPU 450. They areoutput to the PAL 484 via bus 636.

The PIC 482 also outputs the signals BEEP and BEEPCLR* on bus 640 tocontrol the beeper one shot 630.

The PIC 482 also outputs a signal MEM1 (not shown) which is used toswitch pages between the high address section of EPROM 456 and the lowaddress section of battery RAM 458. Two other signals PAGE SEL 0 andPAGE SEL 1 (not shown) are output to select between four 16K pages inEPROM 456.

The four temperature sensors are National LM 135 zener diode typesensors with a zener voltage/temperature dependence of 10 mV/°K. Thezener diodes are driven from the regulated power supply 498 through the20K resistor 496. The current through the zeners varies fromapproximately 560 μA to 615 μA over the 0° C. to 100° C. operatingrange. The zener self heating varies from 1.68 mW to 2.10 mW over thesame range.

The multiplexers 492 and 494 are DG409 analog switches. The voltages onlines 488 and 490 are amplified by an AD625KN instrumentation amplifierwith a transfer function of V_(OUT) =3*V_(IN) -7.5. The A/D converter486 is an AD7672 with an input range from 0-5 volts. With the zenertemperature sensor output from 2.73 to 3.73 volts over the 0° C. to 100°C. range, the output of the amplifier 578 will be 0.69 volts to 3.69volts, which is comfortably within the A/D input range.

The keys to highly accurate system performance are good accuracy and lowdrift with changes in ambient temperature. Both of these goals areachieved by using a precision voltage reference source, i.e.,calibration voltage generator 506, and continuously monitoring itsoutput through the same chain of electronics as are used to monitor theoutputs of the temperature sensors and the AC line voltage on line 510.

The calibration voltage generator 506 outputs two precision voltages onlines 650 and 652. One voltage is 3.75 volts and the other is 3.125volts. These voltages are obtained by dividing down a regulated supplyvoltage using a string of ultralow drift, integrated, thin filmresistors with a 0.05% match between resistors and a 5 ppm/degree C.temperature drift coefficient between resistors. The calibration voltagegenerator also generates -5 volts for the A/D converter referencevoltage and -7.5 volts for the instrumentation amplifier offset. Thesetwo voltages are communicated to the A/D 486 and the amplifier 578 bylines which are not shown. These two negative voltages are generatedusing the same thin film resistor network and OP 27 GZ op-amps (notshown). The gain setting resistors for the operational amplifier 578 arealso the ultralow drift, thin-film, integrated, matched resistors.

The control firmware, control electronics and the block design aredesigned such that well-to-well and instrument-to-instrumenttransportability of PCR protocols is possible.

High throughput laboratories benefit from instruments which are easy touse for a wide spectrum of lab personnel and which require a minimalamount of training. The software for the invention was developed tohandle complex PCR thermocycling protocols while remaining easy toprogram. In addition, it is provided with safeguards to assure theintegrity of samples during power interruptions, and can document thedetailed events of each run in safe memory.

After completing power-up self-checks shown in FIGS. 53 and 54, toassure the operator that the system is operating properly, the userinterface of the invention offers a simple, top-level menu, inviting theuser to run, create or edit a file, or to access a utility function. Noprogramming skills are required, since preexisting default files can bequickly edited with customized times and temperatures, then stored inmemory for later use. A file protection scheme prevents unauthorizedchanges to any user's programs. A file normally consists of a set ofinstructions to hold a desired temperature or to thermocycle. Complexprograms are created by linking files together to form a method. Acommonly used file, such as a 4° C. incubation following a thermocycle,can be stored and then incorporated into methods created by other users.A new type of file, the AUTO file is a PCR cycling program which allowsthe user to specify which of several types of changes to controlparameters will occur each cycle: time incrementing (auto segmentextension, for yield enhancement), time decrementing, or temperatureincrementing or decrementing. For the highest degree of controlprecision and most reliable methods transferability, temperatures aresetable to 0.1° C., and times are programmed to the nearest second. Theinvention has the ability to program a scheduled PAUSE at one or moresetpoints during a run for reagent additions or for removal of tubes atspecific cycles.

The system of the invention has the ability to store a 500 recordhistory file for each run. This feature allows the user to review theindividual steps in each cycle and to flag any special status or errormessages relating to irregularities. With the optional printer, theinvention provides hardcopy documentation of file and method parameters,run-time time/temperature data with a time/date stamp, configurationparameters, and sorted file directories.

In order to assure reproducible thermocycling, the computed sampletemperature is displayed during the ramp and hold segments of eachcycle. A temperature one degree different than the set temperature isnormally used to trigger the ramp-time and hold-time clocks, but thiscan be altered by the user. For some tube types, the invention willprovide the proper time constant for the type of tube and volume used (acapability is provided for users to enter time constants in a table forother tube types, so subsequently only the tube type would have to beentered before a run), so the sample will always approach the desiredsample temperature with the same accuracy, regardless of whether long orshort sample incubation times have been programmed. Users can programslow ramps for the specialized annealing requirements of degenerateprimer pools, or very short (1-5 sec) high-temperature denaturationperiods for very GC rich targets. Intelligent defaults are preprogrammedfor 2- and 3-temperature PCR cycles.

Diagnostic tests can be accessed by any users to check the heating andcooling system status and to verify the calibration, since the softwaregives Pass/Fail reports. In addition, a system performance programperforms a comprehensive subsystem evaluation and generates a summarystatus report.

The control firmware is comprised of several sections which are listedbelow:

--Diagnostics

--Calibration

--Install

--Real time operating system

--Nine prioritized tasks that manage the system

--Start-up sequence

--User interface

The various sections of the firmware will be described with eithertextual description, pseudocode or both. The actual source code in Clanguage is included below as Microfiche Appendix F.

Features of the firmware are:

1. A Control system that manages the average sample block temperature towithin ±0.1° C. as well as maintaining the temperature non-uniformity asbetween wells in the sample block to within ±0.5° C.

2. A temperature control system that measures and compensates for linevoltage fluctuations and electronic temperature drift.

3. Extensive power up diagnostics that determine if system componentsare working.

4. Comprehensive diagnostics in the install program which qualify theheating and cooling systems to insure they are working properly.

5. A logical and organized user interface, employing a menu drivensystem that allows instrument operation with minimal dependency on theoperators manual.

6. The ability to link up to 17 PCR protocols and store them as amethod.

7. The ability to store up to 150 PCR protocols and methods in the userinterface.

8. A history file that records up to 500 events of the previous run aspart of the sequence task.

9. The ability to define the reaction volume and tube size type at thestart of a run for maximum temperature accuracy and control as part ofthe user interface and which modifies tau (the tube time constant) inthe PID task.

10. Upon recovery from a power failure, the system drives the sampleblock to 4° C. to save any samples that may be loaded in the samplecompartment. The analyzer also reports the duration of the power failureas part of the start-up sequence.

11. The ability to print history file contents, "run time" parametersand stored PCR protocol parameters as part of the print task.

12. The ability to configure the temperature to which the apparatus willreturn during any idle state.

13. The ability to check that the setpoint temperature is reached withina reasonable amount of time.

14. The ability to control the instrument remotely via an RS232 port.

There are several levels of diagnostics which are described below:

A series of power-up tests are automatically performed each time theinstrument is turned on. They evaluate critical areas of the hardwarewithout user intervention. Any test that detects a component failurewill be run again. If the test fails twice, an error message isdisplayed and the keyboard is electronically locked to prevent the userfrom continuing.

The following areas are tested:

Programmable Peripheral Interface device

Battery RAM device

Battery RAM checksum

EPROM devices

Programmable Interface Timer devices

Clock/Calendar device

Programmable Interrupt Controller device

Analog to Digital section

Temperature sensors

Verify proper configuration plug

A Series of service only diagnostics are available to final testers atthe manufacturer's location or to field service engineers through a"hidden" keystroke sequence (i.e. unknown to the customer). Many of thetests are the same as the ones in the start up diagnostics with theexception that they can be continually executed up to 99 times.

The following areas are tested:

Programmable Peripheral Interface device

Battery RAM device

Battery RAM checksum

EPROM devices

Programmable Interface Timer devices

Clock/Calendar device

Programmable Interrupt Controller device

Analog to Digital section

RS-232 section

Display section

Keyboard

Beeper

Ramp Cooling Valves

Check for EPROM mismatch

Firmware version level

Battery RAM Checksum and Initialization

Clear Calibration Flag

Heated Cover heater and control circuitry

Edge heater and control circuitry

Manifold heater and control circuitry

Central heater and control circuitry

Sample block thermal cutoff test

Heated cover thermal cutoff test

User diagnostics are also available to allow the user to perform a quickcool and heat ramp verification test, an extensive confirmation of theheating and cooling system and to verify sample block calibration. Thesediagnostics also allow the user to view the history file, which is asequential record of events that occurred in the previous run. Therecords contain time, temperature, setpoint number, cycle number,program number and status messages.

Remote Diagnostics are available to allow control of the system from anexternal computer via the RS-232 port. All user functions and servicediagnostics and instrument calibration can be performed remotely.

Calibration to determine various parameters such as heater resistance,etc. is performed. Access to the calibration screen is limited by a"hidden" key sequence (i.e. unknown to the customer). The followingparameters are calibrated:

The configuration plug is a module that rewires the chiller unit, sampleblock heaters, coolant pump and power supplies for the proper voltageand frequency (100 V/50 Hz, 100/60 Hz, 120/60 Hz, 220/50 Hz or 230/50Hz). The user enters the type of configuration plug installed. Thefirmware uses this information to compute the equivalent resistance ofthe sample block heaters. Upon power-up, the system verifies that theconfiguration plug selected is consistent with the current line voltageand frequency.

The heater resistance must be determined in the calibration process sothat precise calculations of heater power delivered can be made. Theuser enters the actual resistances of the six sample block heaters (twomain heaters, two manifold heaters and two edge heaters). Theconfiguration plug physically wires the heater in series for 220-230 VACand in parallel for 100-120 VAC operation. The firmware computes theequivalent resistance of each of the three heaters by the followingformula:

    For 100-120 VAC: R.sub.eq =(R.sub.1 *R.sub.2)/R.sub.1 +R.sub.2(7)

    For 220-230 VAC: R.sub.eq =R.sub.1 +R.sub.2                (8)

The equivalent resistance is used to deliver a precise amount of heatingpower to the sample block (Power=Voltage² ×Resistance).

The calibration of the A/D circuit is necessary so that temperatures canbe precisely measured. This is performed by measuring two test pointvoltages (TP6 and TP7 on the CPU board) and entering the measuredvoltages. The output of the A/D at each voltage forms the basis of a twopoint calibration curve. These voltages are derived from a 5 voltprecision source and are accurate and temperature independent. At thestart of each run, these voltages are read by the system to measureelectronic drift due to temperature because any changes in A/D output isdue to temperature dependencies in the analog chain (multiplexer, analogamplifier and A/D converter).

Calibration of the four temperature sensors (sample block, ambient,coolant and heated cover) is performed for accurate temperaturemeasurements. Prior to installation into an instrument, the ambient,coolant, and heated cover temperature sensors are placed in a water bathwhere their output is recorded (XX.X °C. at YYYY mV). These values arethen entered into the system. Since temperature accuracy in these areasis not critical, a one point calibration curve is used.

The sample block sensor is calibrated in the instrument. An array of 15accurate temperature probes is strategically placed in the sample blockin the preferred embodiment. The output of the temperature probes iscollected and averaged by a computer. The firmware commands the block togo to 40° C. After a brief stabilizing period the user enters theaverage block temperature as read by the 15 probes. This procedure isrepeated at 95° C., forming a two point calibration curve.

Calibration of the AC to DC line voltage sampling circuit is performedby entering into the system the output of the AC to DC circuit for twogiven AC input voltages, forming a two point calibration curve. Theoutput of the circuit is not linear over the required range (90-260 VAC)and therefore requires two points at each end (100 and 120, 220 and 240VAC), but only uses one set based on the current input voltage.

An accurate measure of AC voltage is necessary to deliver a preciseamount of power to the sample block (Power=Voltage² ×Resistance). TheInstall program is a diagnostic tool that performs an extensive test ofthe cooling and heating systems. Install measures or calculates controlcooling conductance, ramp cooling conductance at 10° C. and 18° C.,cooling power at 10° C. and 20° C., sample block thermal and coolantcapacity and sample block sensor lag. The purpose of install is threefold:

1. To uncover marginal or faulty components.

2. To use some of the measured values as system constants stored inbattery backed up RAM to optimize the control system for a giveninstrument.

3. To measure heating and cooling system degradation over time.

Install is executed once before the system is shipped and should also berun before use or whenever a major component is replaced. The Installprogram may also be run by the user under the user diagnostics.

The heater ping test verifies that the heaters are properly configuredfor the current line voltage (i.e. in parallel for 90-132 VAC and inseries for 208-264 VAC). The firmware supplies a burst of power to thesample block and then monitors the rise in temperature over a 10 secondtime period. If the temperature rise is outside a specified ramp ratewindow, then the heaters are incorrectly wired for the current linevoltage and the install process is terminated.

The control cooling conductance tests measures the thermal conductanceK_(cc) across the sample block to the control cooling passages. Thistest is performed by first driving the sample block temperature to 60°C. (ramp valves are closed), then integrating the heater power requiredto maintain the block at 60° C. over a 90 second time period. Theintegrated power is divided by the sum of the difference between theblock and coolant temperature over the interval.

    K.sub.cc =Σ Heater Power.sub.60° C. /Σ Block-Coolant Temp(9)

Typical values are 1.31 to 1.78 Watts/°K. A low K_(cc) may indicate aclogged liner(s). A high K_(cc) may be due to a ramp valve that is notcompletely closed, leakage of the coolant to the outside diameter of theliner, or a liner that has shifted.

The block thermal capacity (Blk Cp) test measures the thermal capacityof the sample block by first controlling the block at 35° C. thenapplying the maximum power to the heaters for 20 seconds. The blockthermal capacity is equal to the integrated power divided by thedifference in block temperature. To increase accuracy, the effect ofbias cooling power is subtracted from the integrated power.

    Blk Cp=ramp time*(heater-control cool pwr)/delta temp.     (10)

where:

ramp time=20 seconds

heater power=500 watts

control cool=(Z block-coolant temp)*K_(cc)

delta temp=TBlock_(t=20) -TBlock_(t=0)

The typical value of Block Cp is 567 Joules/°K.±45 for a 60 Hzinstrument. Assuming a normal K_(cc) value, an increase in block thermalcapacity is due to an increase in thermal loads, such as moisture in thefoam backing, loss of insulation around the sample block, or a decreasein heater power such as a failure of one of the six heater zones or afailure of the electronic circuitry that drives the heater zones, or anincorrect or an incorrectly wired voltage configuration module.

A chiller test measures the system cooling output in watts at 10° C. and18° C. The system cooling power, or chiller output, at a giventemperature is equal to the summation of thermal loads at thattemperature. The main components are: 1. heating power required tomaintain the block at a given temperature, 2. power dissipated by thepump used to circulate the coolant around the system, and 3. losses inthe coolant lines to the ambient. The chiller power parameter ismeasured by controlling the coolant temperature at either 10° C. or 18°C. and integrating the power applied to the sample block to maintain aconstant coolant temperature, over a 32 second interval. The differencebetween the block and coolant temperature is also integrated to computelosses to ambient temperature.

    Chiller power=Σ Heating power+Pump power+(Kamb*Σ (blk-cool temp))                                                    (11)

where:

heating power=Sum of heating power required to maintain coolant at 10°C. or 18° C. over time 32 seconds.

Pump Power=Circulating pump, 12 watts

Kamb=Conductance to ambient, 20 watts/°C.

blk-cool temp=Sum of difference in block and coolant temp over time 32seconds

The typical value for chiller power is 256 watts±76 for a 60 Hzinstrument at 10° C. and 383 watts±48 at 18° C. Low chiller power may bedue to an obstruction in the fan path, a defective fan, or a marginal orfaulty chiller unit. It may also be due to a miswired voltageconfiguration plug.

A ramp cooling conductance (K_(c)) test measures the thermal conductanceat 10° C. and 18° C. across the sample block to the ramp and controlcooling passages. This test is performed by first controlling thecoolant temperature at 10° C. or 18° C., then integrating, over a 30second time interval, the heating power applied to maintain the coolantat the given temperature divided by the difference of block and coolanttemperature over the time interval.

    K.sub.c =Σ Heating power/Σ (block-coolant temperature)

Typical values for K_(c) are 33.4 watts/°K.±7.4 at 10° C. and 37.6watts/°K.±5 at 18° C. A low K_(c) may be due to a closed or obstructedramp valve, kinked coolant tubing, weak pump or a hard water/Prestone™mixture.

A sensor lag test measures the block sensor lag by first controlling theblock temperature to 35° C. and then applying 500 watts of heater powerfor 2 seconds and measuring the time required for the block to rise 1°C. Typical values are 13 to 15 units, where each unit is equal to 200ms. A slow or long sensor lag can be due to a poor interface between thesensor and the block, such as lack of thermal grease, a poorly machinedsensor cavity or a faulty sensor.

The remaining install tests are currently executed by the installprogram but have limited diagnostic purposes due to the fact that theyare calculated values or are a function of so many variables that theirresults do not determine the source of a problem accurately.

The install program calculates the slope of the ramp cooling conductance(S_(c)) between 18° C. and 10° C. It is a measure of the linearity ofthe conductance curve. It is also used to approximate the ramp coolingconductance at 0° C. Typical values are 0.40±0.2. The spread in valuesattest to the fact that it is just an approximation.

    S.sub.c =(Kc.sub.-- 18°-Kc.sub.-- 10°)/(18° C.-10° C.)                                         (13)

The install program also calculates the cooling conductance K_(c0).K_(c0) is an approximation of the cooling conductance at 0° C. The valueis extrapolated from the actual conductance at 10° C. Typical values are23 watts/°K.±5. The formula used is:

    K.sub.c0 =Kc.sub.-- 10-(Sc*10° C.)                  (14)

Characters enclosed in { } indicate the variable names used in thesource code.

Heater-Ping Test Pseudocode:

The heater ping test verifies that the heaters are properly wired forthe current line voltage.

Get the sample block and coolant to a known and stable point.

Turn ON the ramp cooling valves

Wait for the block and coolant to go below 5° C.

Turn OFF ramp cooling valves

Measure the cooling effect of control cooling by measuring the blocktemperature drop over a 10 second time interval. Wait 10 seconds forstabilization before taking any measurements.

Wait 10 seconds

temp1=block temperature

Wait 10 seconds

temp2 =block temperature

{tempa}=temp2-temp1

Examine the variable {linevolts} which contains the actual measured linevoltage. Pulse the heater with 75 watts for a line voltage greater thanor equal to 190 V or with 300 watts if it less than or equal to 140 V.##STR17## Measure the temperature rise over a 10 second time period. Theresult is the average heat rate in 0.01°/second.

temp1=block temperature

Wait 10 seconds

temp2=block temperature

{tempb}=temp2-temp1

Subtract the average heat rate {tempb} from the control cooling effectto calculate true heating rate

    heat.sub.-- rate={tempb}-{tempa}                           (17)

Evaluate the heat₋₋ rate. For 220 V-230 V, the heat rate should be lessthan 0.30°/second. For 100 V-120 V the heat rate should be greater than0.30°/second. ##STR18## KCC Test Pseudocode:

This test measures the control cooling conductance also known as K_(cc).

K_(cc) is measured at a block temperature of 60° C.

Drive block to 60° C.

Maintain block temperature at 60° C. for 300 seconds

Integrate the power being applied to the sample block heaters over a 90second time period. Measure and integrate the power required to maintainthe block temperature with control cooling bias. ##STR19## Compute theconductance by dividing the power sum by the temperature sum. Note thatthe units are 10 mW/°K.

    K.sub.cc =({main.sub.-- pwr.sub.-- sum}+{aux.sub.-- pwr.sub.-- sum})/{dt.sub.-- sum}                                     (18)

BLOCK CP Test Pseudocode:

This test measures the sample block thermal capacity.

Drive the block to 35° C.

Control block temperature at 35° C. for 5 seconds and record initialtemperature.

    initial.sub.-- temp=block temperature

Deliver maximum power to heaters for 20 seconds while summing thedifference in block to coolant temperature as well as heater power.##STR20## Compute the total joules applied to the block from the mainheater and control cooling. Divide by temp change over the interval tocompute thermal capacity.

    Block CP=ramptime*(heater power-cool.sub.-- joule)/delta.sub.-- temp(21)

where:

ramptime=20 seconds

heater power=500 Watts

COOL₋₋ PWR₋₋ 10:

This test measures the chiller power at 10° C.

Control the coolant temperature at 10° C. and stabilize for 120 secs.##STR21## At this point, the coolant has been at 10° C. for 120 secondsand has stabilized. Integrate, over 32 seconds, the power being appliedto maintain a coolant temperature of 10° C. ##STR22## Compute the numberof joules of energy added to the coolant mass during the integrationinterval. "(coolant temp-cool₋₋ init)" is the change in coolant tempduring the integration interval. 550 is the Cp of the coolant in joules,thus the product is in joules. It represents the extra heat added to thecoolant which made it drift from setpoint during the integrationinterval. This error is subtracted below from the total heat appliedbefore calculating the cooling power.

    cool.sub.-- init=(coolant temp-cool.sub.-- init)*550J      (22)

Add the main power sum to the aux heater sum to get joules dissipated in32 seconds. Divide by 32 to get the average joules/sec.

    {main.sub.-- pwr.sub.-- sum}=({main.sub.-- pwr.sub.-- sum}+{aux.sub.-- pwr.sub.-- sum}-cool.sub.-- init)/32                      (23)

Compute the chiller power at 10° C. by summing all the chiller powercomponents.

    Power.sub.10°C. =main.sub.-- power.sub.-- sum+PUMP PWR+(K.sub.-- AMB*delta.sub.-- temp.sub.-- sum)                         (24)

where:

{main₋₋ pwr₋₋ sum}=summation of heater power over interval

PUMP PWR=12 Watts, pump that circulates coolant

delta₋₋ temp₋₋ sum=summation of amb-coolant over interval

K₋₋ AMB=20 Watts/K, thermal conductance from cooling to ambient.

KC₋₋ 10 Test Pseudocode:

This test measures the ramp cooling conductance at 10° C.

Control the coolant temperature at 10° C.±0.5 and allow it to stabilizefor 10 seconds.

At this point, the coolant is at setpoint and is being controlled.Integrate, over a 90 second time interval, the power being applied tothe heaters to maintain the coolant at 10° C. Sum the difference betweenthe block and coolant temperatures. ##STR23## Compute the energy injoules delivered to the block over the summation period. Units are in0.1 watts.

    {main.sub.-- pwr.sub.-- sum}={main.sub.-- pwr.sub.-- sum}+{aux.sub.-- pwr.sub.-- sum}                                           (25)

Divide the power sum by block-coolant temperature sum to get rampcooling conductance in 100 mW/K.

    Kc.sub.-- 10={main.sub.-- pwr.sub.-- sum}/{dt.sub.-- sum}  (26)

COOL₋₋ PWR₋₋ 18 Test Pseudocode:

This test measures the chiller power at 18° C.

Get the sample block and coolant to a known and stable point. Controlthe coolant temperature at 18° C. and stabilize for 120 secs. ##STR24##

At this point the coolant has been at 18° C. for 120 seconds and hasstabilized. Integrate, over 32 seconds, the power being applied tomaintain a coolant temperature of 18° C. ##STR25##

Compute the number of joules of energy added to the coolant mass duringthe integration interval. "(coolant temp-cool₋₋ init)" is the change incoolant temp during the integration interval. 550 is the Cp of thecoolant in joules, thus the product is in joules. It represents theextra heat added to the coolant which made it drift setpoint during theintegration interval. This error is subtracted below from the total heatapplied before calculating the cooling power.

    cool.sub.-- init=(coolant temp-cool.sub.-- init)*550J      (27)

Add main power sum to aux heater sum to get joules dissipated in 32seconds. Divide by 32 to get the average joules/sec.

    {main.sub.-- pwr.sub.-- sum}=({main.sub.-- pwr.sub.-- sum}+{aux.sub.-- pwr.sub.-- sum}-cool.sub.-- init)/32                      (28)

Compute the chiller power at 18° C. by summing all the chiller powercomponents.

    Power.sub.18°C. =main.sub.-- power.sub.-- sum+PUMP PWR+(K.sub.-- AMB*delta.sub.-- temp.sub.-- sum)                         (29)

where:

{main₋₋ pwr₋₋ sum}=summation of heater power over interval

PUMP PWR=12 Watts, pump that circulates coolant

delta₋₋ temp₋₋ sum=summation of amb-coolant over interval

K₋₋ AMB=20 Watts/K, Thermal conductance from cooling to ambient.

KC₋₋ 18 Test Pseudocode:

This test measures the ramp cooling conductance at 18° C.

Control the coolant temperature at 18° C.±0.5 and allow it to stabilizefor 10 seconds.

At this point, the coolant is at setpoint and being controlled.Integrate, over a 90 second time interval, the power being applied tothe heaters to maintain the coolant at 18° C. Sum the difference betweenthe block and coolant temperature. ##STR26##

Compute the energy in joules delivered to the block over the summationperiod. Units are in 0.1 watts.

    {main.sub.-- pwr.sub.-- sum}={main.sub.-- pwr.sub.-- sum}+{aux.sub.-- pwr.sub.-- sum}                                           (30)

Divide power sum by block-coolant temperature sum to get ramp coolingconductance in 100 mW/K.

    Kc.sub.-- 18={main.sub.-- pwr.sub.-- sum}/{dt.sub.-- sum}  (31)

SENLAG Test Pseudocode:

This test measures the sample block sensor lag.

Drive the block to 35° C. Hold within ±0.2° C. for 20 seconds thenrecord temperature of block.

    {tempa}=block temperature

Deliver 500 watts of power to sample block.

Apply 500 watts of power for the next 2 seconds and count the amount ofiterations through the loop for the block temperature to increase 1° C.Each loop iteration executes every 200 ms, therefore actual sensor lagis equal to count*200 ms. ##STR27##

REAL TIME OPERATING SYSTEM - CRETIN

CRETIN is a stand alone, multitasking kernel that provides systemservices to other software modules called tasks. Tasks are written inthe "C" language with some time critical areas written in Intel 8085assembler. Each task has a priority level and provides an independentfunction. CRETIN resides in low memory and runs after the startupdiagnostics have successfully been executed.

CRETIN handles the task scheduling and allows only one task to run at atime. CRETIN receives all hardware interrupts thus enabling waitingtasks to run when the proper interrupt is received. CRETIN provides areal time clock to allow tasks to wait for timed events or pause forknown intervals. CRETIN also provides intertask communication through asystem of message nodes.

The firmware is composed of nine tasks which are briefly described inpriority order below. Subsequent sections will describe each task ingreater detail.

1. The control task (PID) is responsible for controlling the sampleblock temperature.

2. The keyboard task is responsible for processing keyboard input fromthe keypad.

3. The timer task waits for a half second hardware interrupt, then sendsa wake up message to both the sequence and the display task.

4. The sequence task executes the user programs.

5. The pause task handles programmed and keypad pauses when a program isrunning.

6. The display task updates the display in real time.

7. The printer task handles the RS-232 port communication and printing.

8. The LED task is responsible for driving the heating LED. It is alsoused to control the coolant temperature while executing Install.

9. The link task starts files that are linked together in a method bysimulating a keystroke.

Block Temperature Control Proqram (PID Task)

The Proportional Integral Differential (PID) task is responsible forcontrolling the absolute sample block temperature to 0.1° C., as well ascontrolling the sample block temperature non-uniformity (TNU, defined asthe temperature of the hottest well minus the temperature of the coldestwell) to less than ±0.5° C. by applying more heating power to theperimeter of the block to compensate for losses through the guard bandedges. The PID task is also responsible for controlling the temperatureof the heated cover to a less accurate degree. This task runs 5 timesper second and has the highest priority.

The amount of heating or cooling power delivered to the sample block isderived from the difference or "error" between the user specified sampletemperature stored in memory, called the setpoint, and the currentcalculated sample temperature. This scheme follows the standard loopcontrol practice. In addition to a power contribution to the filmheaters directly proportional to the current error, i.e., theproportional component, (setpoint temperature minus sample blocktemperature), the calculated power also incorporates an integral termthat serves to close out any static error (Setpoint temperature-Blocktemperature less than 0.5° C.). This component is called the integralcomponent. To avoid integral term accumulation or "wind-up",contributions to the integral are restricted to a small band around thesetpoint temperature. The proportional and integral component gains havebeen carefully selected and tested, as the time constants associatedwith the block sensor and sample tube severely restrict the system'sphase margin, thus creating a potential for loop instabilities. Theproportional term gain is P in Equation (46) below and the integral termgain is Ki in Equation (48) below.

The PID task uses a "controlled overshoot algorithm" where the blocktemperature often overshoots its final steady state value in order forthe sample temperature to arrive at its desired temperature as rapidlyas possible. The use of the overshoot algorithm causes the blocktemperature to overshoot in a controlled manner but does not cause thesample temperature to overshoot. This saves power and is believed to benew in PCR instrumentation. A controlled undershoot is also used. Theblock temperature is controlled such that it does not undershoot orovershoot by more than 0.5° C.

The total power delivered to all heater of the sample block to achieve adesired ramp rate is given by:

    Power=(CP/ramp.sub.-- rate)+bias                           (40)

where:

CP=Thermal mass of block

bias=bias or control cooling power

ramp₋₋ rate=T_(final) -T_(initial) /desired ramp rate

This power is clamped to a maximum of 500 watts of heating power forsafety.

With every iteration of the task (every 200 ms) the system appliesheating or ramp cooling power (if necessary) based on the followingalgorithms.

The control system is driven by the calculated sample temperature. Thesample temperature is defined as the average temperature of the liquidin a thin walled plastic sample tube placed in one of the wells of thesample block (hereafter the "block"). The time constant of the system(sample tube and its contents) is a function of the tube type andvolume. At the start of a run, the user enters the tube type and theamount of reaction volume. The system computes a resultant time constant(τ or tau). For the MicroAmp™ tube and 100 microliters of reactionvolume, tau is approximately 9 seconds.

    T.sub.blk-new =T.sub.blk +Power * (200ms/CP)               (41)

    T.sub.samp-new =T.sub.samp +(T.sub.blk-new -T.sub.samp) * 200 ms / tau (42)

where:

T_(blk-new) =Current block temperature

T_(blk) =Block temperature 200 ms ago

Power=Power applied to block

CP=Thermal mass of block

T_(samp-new) =Current sample temperature

T_(samp) =Sample temperature 200 ms ago

tau=Thermal Time Constant of sample tube, adjusted for sensor lag(approximately 1.5)

The error signal or temperature is simply:

    error=Setpoint--T.sub.samp-new                             (43)

As in any closed loop system, a corrective action (heating or coolingpower) is applied to close out part of the current error. In Equation(45) below,F is the fraction of the error signal to be closed out in onesample period (200 mS).

    T.sub.samp-new =T.sub.samp +F* (SP-T.sub.samp)             (44)

where SP=the user setpoint temperature

Due to the large lag in the system (long tube time constant), thefraction F is set low.

Combining formulas (42) and (44) yields:

    T.sub.samp-new =T.sub.samp +(T.sub.blk-new -T.sub.samp) * .2/tau=T.sub.samp +F* (SP=T.sub.samp)                                       (45)

Combining formulas (41) and (45) and adding a term P (the proportionalterm gain) to limit block temperature oscillations and improve systemstability yields:

    Pwr=CP * P/T * ((SP-T.sub.samp) * F * tau/T+T.sub.samp -T.sub.blk) (46)

where

P=the proportional term gain and

T=the sample period of 0.2 seconds (200 msec). and

P/T=1 in the preferred embodiment

Equation (46) is a theoretical equation which gives the power (Pwr)needed to move the block temperature to some desired value withoutaccounting for losses to the ambient through the guardbands, etc.

Once the power needed to drive the block is determined via Equation(46), this power is divided up into the power to be delivered to each ofthe three heater zones by the areas of these zones. Then the losses tothe manifolds are determined and a power term having a magnitudesufficient to compensate for these losses is added to the amount ofpower to be delivered to the manifold heater zone. Likewise, anotherpower term sufficient to compensate for power lost to the block supportpins, the block temperature sensor and the ambient is added to the powerto be delivered to the edge heater zones. These additional terms and thedivision of power by the area of the zones convert Equation (46) toEquations (3), (4) and (5) given above.

Equation (46) is the formula used by the preferred embodiment of thecontrol system to determine the required heating or cooling power to thesample block.

When the computed sample temperature is within the "integral band" i.e.,±0.5° C. around the target temperature (SP), the gain of theproportional term is too small to close out the remaining error.Therefore an integral term is added to the proportional term to closeout small errors. The integral term is disabled outside the integralband to prevent a large error signal from accumulating. The algorithminside the "integral band" is as follows:

    Int.sub.--i sum (new)=Int.sub.-- sum (old)+(SP-T.sub.samp) (47)

    pwr.sub.-- adj=ki * Int.sub.-- sum (new)                   (48)

where,

Int₋₋ sum=the sum of the sample period of the difference between the SPand T_(samp) temperature, and

Ki=the integral gain (512) in the preferred embodiment).

Once a heating power has been calculated, the control softwaredistributes the power to the three film heater zones 254, 262 and 256 inFIG. 13 based on area in the preferred embodiment. The edge heatersreceive additional power based upon the difference between the blocktemperature and ambient temperature. Similarly, the manifold heatersreceive additional power based upon the difference between the blocktemperature and the coolant temperature.

Characters enclosed in {} in the pseudocode given below for the PID taskcorrespond to the variable names used in the source code of MicroficheAppendix F.

PID Pseudocode

Upon System Power up or Reset

Initialize PID variables

Read the line frequency

Initialize PIT and system clock

Turn off ramp cooling

Turn off all heaters

Calculate heater resistances

Do Forever--executes every 200 ms

If (block temperature>105) then

Turn off heaters

Turn on ramp valves

Display error message

Read the line voltage {linevolts}

Read the coolant sensor and convert to temperature {h2otemp}

Check if sensor reading is within normal operating range

Display an error message if it is not.

Read the ambient sensor and convert to temperature {ambtemp}

Check if sensor reading is within normal operating range

Display an error message if it is not.

Read the heated cover sensor and convert to temperature {cvrtemp}

Check if sensor reading is within normal operating range

Display an error message if it is not.

Read the sample block sensor and convert to temperature {blktemp}

Check if sensor reading is within normal operating range

Display an error message if it is not.

This portion of the code also reads the temperature stable voltagereference and compares the voltage to a reference voltage that wasdetermined during calibration of the instrument. If there is anydiscrepancy, the electronics have drifted and the voltage readings fromthe temperature sensors are adjusted accordingly to obtain accuratetemperature readings.

Compute the sample temperature {tubetenths} or the temperature that getsdisplayed by using a low-pass digital filter.

    tubetenths=TT.sub.n-1 +(TB.sub.n -TT.sub.n-1) * T/tau      (49)

where TT_(n-1) =last sample temp {tubetenths}

TB_(n) =current block sensor temp {blktenths}

T=sample interval in seconds=200 ms

tau=tau tube {cf₋₋ tau}--tau sensor {cf₋₋ lag}

Equation (49) represents the first terms of a Taylor series expansion ofthe exponential that defines the calculated sample temperature given asEquation (6) above.

Compute the temperature of the foam backing underneath the sample block,{phantenths} known as the phantom mass. The temperature of the phantommass is used to adjust the power delivered to the block to account forheat flow in and out of the phantom mass. The temperature is computed byusing a low pass digital filter implemented in software.

    phantenths=TT.sub.n-1 +(TB.sub.n -TT.sub.n-1) * T/tau      (50)

where TT_(n-1) =Last phantom mass temp {phantenths}

TB_(n) =Current block sensor temp {blktenths}

T=Sample interval in seconds=200 ms

tau_(foam) =Tau of foam block=30 secs.

Compute the sample temperature error (the difference between the sampletemperature and the setpoint temperature) {abs₋₋ tube₋₋ err}.

Determine ramp direction {fast₋₋ ramp}=UP₋₋ RAMP or DN₋₋ RAMP

If (sample temperature is within ERR of setpoint (SP)) then PID not infast transition mode. {fast₋₋ ramp}=OFF

where ERR=the temperature width of the "integral band", i.e., the errorband surrounding the target or setpoint temperature.

Calculate current control cooling power {cool₋₋ ctrl} to determine howmuch heat is being lost to the bias cooling channels.

Calculate current ramp cooling power {cool₋₋ ramp}

Calculate {cool₋₋ brkpt}. {cool₋₋ brkpt} is a cooling power that is usedto determine when to make a transition from ramp to control cooling ondownward ramps. It is a function of block and coolant temperature.

The control cooling power {cool₋₋ ctrl} and the ramp cooling power{cool₋₋ ramp} are all factors which the CPU must know to controldownward temperature ramps, i.e., to calculate how long to keep the rampcooling solenoid operated valves open. The control cooling power isequal to a constant plus the temperature of the coolant times thethermal conductance from the block to the bias cooling channels.Likewise, the ramp cooling power is equal to the difference between theblock temperature and the coolant temperature times the thermalconductance from the block to the ramp cooling channels. The coolingbreakpoint is equal to a constant (given in Microfiche Appendix F) timesthe difference in temperature between the block and the coolant.

Calculate a heating or cooling power {int₋₋ pwr} needed to move theblock temperature from its current temperature to the desired setpoint(SP) temperature.

    {int.sub.-- pwr}=KP * CP * [(SP-T.sub.samp) * {cf.sub.-- kd}+Ts-T.sub.BLK]( 51)

where:

KP=Proportional gain=P/T in Equation (46)=approximately one in thepreferred embodiment

CP=Thermal mass of block

SP=Temperature setpoint

T_(SAMP) =Sample temperature

T_(BLK) =Block temperature

cf₋₋ kd=Tau * K_(d) / Delta₋₋ t where tau is the same tau as used inEquation (49) and K_(d) is a constant given in Microfiche Appendix F andDelta₋₋ t is the 200 msec sample period.

If the flag {normal₋₋ power} is set to 0 when doing a down ramp, thenuse the following power equation:

    CP * P/t.sub.interval * ((SP-T.sub.B.sbsb.n-1) * F * tau/t.sub.interval) (51.1)

If (sample temperature is within {cf₋₋ iband} of setpoint) then

integrate sample error {i₋₋ sum} else

    clear {i.sub.-- sum=0}.                                    (52)

Calculate the integral term power.

    integral term={i.sub.-- sum} * constant {cf.sub.-- term}.  (53)

Add the integral term to the power.

    {int.sub.-- pwr}={int.sub.-- pwr}+integral term            (54)

Adjust power to compensate for heating load due to the effects of thephantom mass (foam backing) by first finding the phantom mass power thenadding it to power {int₋₋ pwr}.

Calculate phantom mass power {phant₋₋ pwr} by: ##STR28##

Compute power needed in manifold heaters {auxl₋₋ power} which willcompensate for loss from the sample block into the manifold edges thathave coolant flowing through it. Note that if the system is in adownward ramp, {aux1₋₋ power}=0. The manifold zone power required isdescribed below:

    {aux1.sub.-- power}=K1*(T.sub.BLK -T.sub.AMB)+K2*(T.sub.BLK -T.sub.COOL)+K5*(dT/dt)                                   (57)

where:

K1=Coefficient {cf₋₋ 1coeff}

K2=Coefficient {cf₋₋ 2coeff}

K5=Coefficient {cf₋₋ 5coeff}

dT/dt=Ramp rate

T_(BLK) =Block temperature

T_(AMB) =Ambient temperature

T_(COOL) =Coolant temperature

Compute power needed in edge heaters {aux2₋₋ power} which willcompensate for losses from the edges of the sample block to ambient.Note that if we are in a downward ramp {aux2₋₋ power}=0. The edge zonepower required is described below:

    {aux2.sub.-- power}=K3*(T.sub.BLK -T.sub.AMB)+K4*(T.sub.BLK -T.sub.COOL)+K6*(dT/dt)                                   (58)

where:

K3=Coefficient {cf₋₋ 3coeff}

K4=Coefficient {cf₋₋ 4coeff}

K6=Coefficient {cf₋₋ 6coeff}dT/dt=Ramp rate

T_(BLK) =Block temperature

T_(AMP) =Ambient temperature

T_(COOL) =Coolant temperature

Delete contribution of manifold {aux1₋₋ power} and edge heater power{aux2₋₋ power} to obtain total power that must be supplied by mainheaters and coolers.

    {int.sub.-- pwr}={int.sub.- pwr}--{aux1.sub.-- power}-{aux2.sub.-- power}(59)

[In an alternate version, the equation {int₋₋ pwr}={int₋₋ pwr}+{aux₋₋power}+{aux2₋₋ power} is used ]

Decide if the ramp cooling should be applied. Note that {cool₋₋ brkpt}is used as a breakpoint from ramp cooling to control cooling.

If (int₋₋ pwr <--cool₋₋ brkpt and performing downward ramp) to decidewhether block temperature is so much higher than the setpointtemperature that ramp cooling is needed then

Turn ON ramp valves else

Turn OFF ramp valves and depend upon bias cooling

If (ramping down)

Check if the gain needs to be adjusted

Check if the alternate power equation (51.1) should be used

At this point, {int₋₋ pwr} contains the total heater power and {aux1₋₋power} and {aux2₋₋ power} contain the loss from the block out to theedges. The power supplied to the auxiliary heaters is composed of twocomponents: aux₋₋ power and int₋₋ power. The power is distributed {int₋₋pwr} to the main and auxiliary heaters based on area.

total₋₋ pwr=int₋₋ pwr

int₋₋ pwr=total₋₋ pwr * 66%

aux1₋₋ power=total₋₋ pwr * 20%+aux1₋₋ power

aux2₋₋ power=total₋₋ pwr * 14%+aux2₋₋ power

If (ramping down)

Depending on the setpoint, the coolant temperature and the time constantof the tube, apply power to the heaters until the ramp coolingterminates.

Compute the number of half cycles for the triac to conduct for each endzone and each iteration of the control loop to send the appropriateamount of power to the heaters. This loop executes once every 1/5second, therefore there are 120/5=24 half cycles at 60 Hz or 100/5=20 at50 Hz. The number of half cycles is a function of requested power {int₋₋pwr}, the current line voltage {linevolts} and the heater resistance.Since the exact power needed may not be delivered each loop, a remainderis calculated {delta₋₋ power} to keep track of what to include from thelast loop.

    int.sub.-- pwr=int.sub.-- pwr+delta.sub.-- power           (60)

Calculate the number of 1/2 cycles to keep the triac on. Index is equalto the number of cycles to keep the triac on.

(61) index=power * main heater ohms * [20 or 24]/ linevolts squaredwhere Equation (61) is performed once for each heater zone and where"power"=int₋₋ pwr for the main heater zone, aux1₋₋ pwr for the manifoldheater zone and aux2₋₋ pwr for the edge heater zone. (61)

Calculate the amount of actual power delivered.

(62) actual₋₋ power=linevolts squared * index / main heater resistance

Calculate the remainder to be added next time.

delta₋₋ power=int₋₋ pwr--actual₋₋ power (63)

Calculate the number of 1/2 cycles for the edge and manifold heatersusing the same technique described for the main heaters by substituting{aux1₋₋ pwr} and {aux2₋₋ pwr} into Equation (60).

Load the calculated counts into the counters that control the main,manifold and edge triacs.

Look at heated cover sensor. If heated cover is less than 105° C., thenload heated cover counter to supply 50 watts of power.

Look at sample temperature. If it is greater than 50° C., turn on HOTLED to warn user not to touch block.

END OF FOREVER LOOP

Keyboard Task

The purpose of the keyboard task is to wait for the user to press a keyon the keypad, compare the key to a list of valid keystrokes for thecurrent state, execute the command function associated with the validkey and change to a new state. Invalid keystrokes are indicated with abeep and then ignored. This task is the heart of the state driven userinterface. It is "state driven" because the action taken depends on thecurrent state of the user interface.

Keyboard Task Pseudocode:

Initialize keyboard task variables.

Turn off the cursor.

Check if the instrument has been calibrated, installed and has thecorrect configuration plug installed

If (the power failed while the user was running a program) then

Compute and display the number of minutes the power was off for.

Write a power failure status record to the history file.

Send a message to the sequence task to start a 4° C. soak.

Give the user the option of reviewing the history file.

If (the user request to review the history file) then

Go to the history file display.

Send a message to pid task to turn on the heated cover.

Display the top level screen.

Do Forever

Send a message to the system that this task is waiting for a hardwareinterrupt from the keypad.

Go to sleep until this interrupt is received.

When awakened, read and decode the key from the keypad.

Get a list of the valid keys for the current state.

Compare the key to the list of valid keys.

If (the key is valid for this state) then

Get the "action" and next state information for this key.

Execute the "action" (a command function) for this state.

Go to the next state.

Else

Beep the beeper for an invalid key.

End of Forever Loop

Timer Task Overview

The purpose of the timer task is to wake up the sequence and the realtime display task every half a second. The timer task asks the system(CRETIN) to wake it up whenever the half second hardware interrupt thatis generated by the clock/calendar device is received. The timer taskthen in turn sends a wake up message to the sequence task and the realtime display task respectively. This intermediate task is necessarysince CRETIN will only service one task per interrupt and thus only thehigher priority task (the sequence task) would execute.

Timer Task Pseudocode:

Do Forever

Send a message to the system that this task is waiting for a hardwareinterrupt from the clock/calendar device.

Go to sleep until this interrupt is received.

When awakened, send a message to the sequence and to the real timedisplay task.

End Forever Loop

Sequence Task Overview

The purpose of the sequence task is to execute the contents of a userdefined program. It sequentially steps through each setpoint in a cycle,consisting of a ramp and a hold segment, and sends out setpointtemperature messages to the pid task which in turn controls thetemperature of the sample block. At the end of each segment, it sends amessage to the real time display task to switch the display and amessage to the printer task to print the segment's runtime information.The user can pause a running program by pressing the PAUSE key on thekeypad then resume the program by pressing the START key. The user canprematurely abort a program by pressing the STOP key. This task executesevery half a second when it is awakened by the timer task.

Sequence Task Pseudocode

Do Forever

Initialize sequence task variables. Wait for a message from the keyboardtask that the user has pressed the START key or selected START from themenu or a message from link task that the next program in a method isready to run.

Go to sleep until this message is received.

When awaken, update the ADC calibration readings to account for anydrift in the analog circuitry.

If (not starting the 4° C. power failure soak sequence) then

Send a message to the printer task to print the PE title line, systemtime and date, program configuration parameters, the program type andits number.

If (starting a HOLD program) then

Get the temperature to hold at {hold₋₋ tp}.

Get the number of seconds to hold for {hold₋₋ time}.

If (ramping down more than 3° C. and {hold₋₋ tp}>45° C.) then

Post an intermediate setpoint.

Else

Post the final setpoint {hold₋₋ tp}.

While (counting down the hold time {hold₋₋ time})

Wait for half second wake up message from timer task.

Check block sensor for open or short.

If (keyboard task detected a PAUSE key) then

Post a setpoint of current sample temp.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Post pre-pause setpoint.

If (an intermediate setpoint was posted) then

Post the final setpoint.

If (the setpoint temp is below ambient temp and will be there for morethan 4 min.) then Set a flag to tell pid task to turn off the heatedcover.

Increment the half second hold time counter {store₋₋ time}.

Post the final setpoint again in case the hold time expired before theintermediate setpoint was reached--this insures the correct setpointwill be written the history file. Write a data record to the historyfile. Send a message to the printer task to print the HOLD info. End ofHOLD program

Else if (starting a CYCLE program) then

Add up the total number of seconds in a cycle {secs₋₋ in₋₋ run}, takinginto account the instrument ramp time and the user programmed ramp andhold times.

Get the total number of seconds in the program by multiplying the numberof seconds in a cycle by the number of cycles in a program {num₋₋ cyc}.

Total {secs₋₋ in₋₋ run}={secs₋₋ in₋₋ run} per cycle * {num₋₋ cyc}. While(counting down the number of cycles {num₋₋ cyc})

While (counting down the number of setpoints {num₋₋ seg})

Get the ramp time {ramp₋₋ time}.

Get the final setpoint temp {t₋₋ final}.

Initialize the ramp variables.

Get the hold time {local₋₋ time}.

Send a message to the real time display task to display the ramp segmentinformation.

Calculate the maximum amount of time it should take to ramp to thesetpoint.

If (the user programmed a ramp time) then

Compute the error {ramp₋₋ err} between the programmed ramp time and theactual ramp time as follows. This equation is based on empirical data.

{ramp₋₋ err}=prog ramp₋₋ rate * 15+0.5 (up ramp)

{ramp₋₋ err}=prog ramp₋₋ rate * 6+1.0 (down ramp)

where:

prog ramp₋₋ rate=(abs(T_(f) -T_(c))-1) / {ramp₋₋ time}

T_(f) =setpoint temp {t₋₋ final}

T_(c) =current block temp {blktemp}

abs=absolute value of the expression

Note: the `--1` is there because the clock starts within 1° C. ofsetpoint.

new ramp₋₋ time=old {ramp₋₋ time}--{ramp₋₋ err}

If (new ramp₋₋ time>old {ramp₋₋ time}) then

new ramp₋₋ time=old {ramp₋₋ time}.

Else

new ramp₋₋ time=0.

While (sample temp is not within a user configured temp {cf₋₋ clk₋₋ dev}of setpoint)

Wait for half second wake up message from timer task.

Post a new ramp setpoint every second.

Else if (ramping down more than 3° C. and {t₋₋ final}>45° C.) then

Post an intermediate setpoint.

While (sample temp is more than 1° C. of setpoint)

Wait for half second wake up message from timer task.

Increment the half second ramp time counter.

Check if the maximum time to ramp to setpoint has expired.

Display an error message if so.

If ramping up and within some integral band of setpoint, change thegain.

If (keyboard task detected a PAUSE key) then

Post a setpoint of current sample temp.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Post pre-pause setpoint.

Post the final setpoint.

While (sample temp is not within a user configured temp

{cf_(--clk) ₋₋ der} of setpoint)

Wait for half second wake up message from timer task.

Increment the half second ramp time counter.

Check if the maximum time to ramp to setpoint has expired.

Display an error message if so.

Check if the gain needs to be changed.

If (keyboard task detected a PAUSE key) then

Post a setpoint of current sample temp.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Post pre-pause setpoint.

Send a message to the printer task to print the ramp information.

Beep beeper to signal end of ramp segment.

Send a message to the real time display task to display the hold segmentinformation.

While (counting down the hold time)

Wait for half second wake up message from timer task.

Increment the half second hold time counter.

If (ramping down and on the upramp part of the block temperatureundershoot and both the block and sample temperature are within 0.2° C.of setpoint) then

Set a flag so that the power equation in pid will revert back to normal(51)

If (keyboard task detected a PAUSE key) then

Post a setpoint of current sample temp.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Post pre-pause setpoint.

Write a data record to the history file.

Send a message to the printer task to print the hold information.

If (the final setpoint temp has drifted more than the

user configurable amount {cf₋₋ temp₋₋ dev}) then

Write an error record to the history file.

Check for a programmed pause and execute if necessary.

Go to next segment.

Send a message to the printer task to print an end of cycle message.

Go to next cycle.

End of CYCLE program.

Else if (starting an AUTO-CYCLE program) then

Add up the total number of seconds in each program {secs₋₋ in₋₋ run}taking into account the instrument ramp time and the user programmedhold times and temperatures which can be automatically incremented ordecremented by a programmed amount, either linearly or geometrically,each cycle.

While (counting down the number of cycles {num₋₋ cyc})

While (counting down the number of setpoints {num₋₋ seg})

Get the final setpoint temp {t₋₋ final}.

Get the hold time {time₋₋ hold}.

Send a message to real time display task to display the ramp segmentinformation.

Check if the user programmed an auto increment or decrement of thesetpoint temp and/or the hold time and adjust them accordingly.

If (the auto increment or decrement of the temp causes the setpoint togo below 0° C. or above 99.9° C.) then

An error record is written to the history file.

The setpoint is capped at either 0° C. or 99.9° C.

If (the auto decrement of the hold time causes the hold time to go below0 seconds) then

An error record is written to the history file.

The hold time is capped at 0° C.

Initialize the ramp variable.

If (ramping down more than 3° C. and {t₋₋ final}>45° C.) then

Post an intermediate setpoint.

While (sample temp is not within 1° C. of setpoint)

Wait for half second wake up message from timer task.

Increment the half second ramp time counter.

If (keyboard task detected a PAUSE key) then

Post a setpoint of current sample temp.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Post pre-pause setpoint.

Check if the maximum amount of time to reach setpoint has expired andwrite an error message to the history file if it has.

Check if the gain needs to be changed.

Post the final setpoint.

While (sample temp is not within a user configured temp

{cf₋₋ clk₋₋ dev} of setpoint)

Wait for half second wake up message from timer task.

Increment the half second ramp time counter.

Check block sensor for open or short.

If (keyboard task detected a PAUSE key) then

Post a setpoint of current sample temp.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Post pre-pause setpoint.

Send a message to the printer task to print the ramp segmentinformation.

Beep beeper to signal end of ramp portion of segment.

Send a message to the real time display task to display the hold segmentinformation.

While (counting down the hold time)

Wait for half second wake up message from timer task.

Increment the half second hold time counter.

If (keyboard task detected a PAUSE key) then

Post a setpoint of current sample temp.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Post pre-pause setpoint.

If (ramping down and on the upramp part of the block temperatureundershoot and both the block and sample temperature are within 0.2° C.of setpoint) then

Set a flag so that the power equation in pid

will revert back to normal (51).

Write a data record to the history file.

Send a message to the printer task to print the hold information.

If (the final setpoint temp has drifted more than the

user configurable amount {cf₋₋ temp₋₋ dev}) then

Write an error record to the history file.

Go to next segment.

Send a message to the printer task to print an end of cycle message.

Go to next cycle.

End of AUTO-CYCLE program.

Else if (starting a POWER FAILURE sequence) then

Post a setpoint of 4° C.

Set a flag {subamb₋₋ hold} so that the pid task will shut off the heatedcover.

DO FOREVER

Wait for a half second wake up message from the timer task.

Increment the half second hold time counter.

END FOREVER LOOP

End of power failure sequence

Write a run end status record to the history file.

If (running a method)

Set a flag {weird₋₋ flag} so the link task will know to send a messageto the sequence task to start the next program running.

Else

Return user interface to idle state display.

End of Forever Loop

Pause Task Overview

The purpose of the pause task is to handle either a pause that the userprograms in a CYCLE or an AUTO-CYCLE program or a pause when the userpresses the PAUSE key on the keypad.

When the sequence task encounters a programmed pause while executing aprogram, it goes to sleep and awakens the pause task. The pause task inturn sends a message to the real time display task to continuallydisplay and decrement the time the user asked to pause for. When thepause timer times out, the pause task sends a message to awaken thesequence task and then goes to sleep. The user can prematurely resumethe program by pressing the START key on the keypad or can prematurelyabort the program by pressing the STOP key.

When the keyboard task detects a PAUSE key while a program is running,it sets a flag {pause₋₋ flag} then waits for the sequence task toacknowledge it. When the sequence task sees this flag set, it sends anacknowledgment message back to the keyboard task then puts itself tosleep. When the keyboard task receives this message, it awakens thepause task. The pause task sends a message to the real time display taskto continually display and increment the amount of time the program ispaused for. The timer will time out when it reaches the pause time limitset by the user in the configuration section. The user can resume theprogram by pressing the START key on the keypad or abort the program bypressing the STOP key.

Pause Task Pseudocode:

Do Forever

Wait for a message from the keyboard task indicating a keypad pause, ora message form the sequence task indicating a user programmed pause.

Go to sleep until a message is received.

When awakened, check a flag for the type of pause initiated.

If (it is a programmed pause) then

Send a message to the real time display task to display the pause timercounting up.

Else

Send a message to the real time display task to display the pause timercounting down.

While (counting down the time out counter)

Send a message to the system to suspend this task for half a second.

Send a message to the printer task to print the pause information.

If (it is a programmed pause) then

Write a status record to the history file.

The pause has timed out so send a message to the wake up the sequencetask.

Send a message to the real time display task to halt the pause display.

Send a message to the real time display task to resume the runningprogram display.

Else (it is a keypad pause)

The pause has timed out and the program must be aborted so send amessage to the system to halt the sequence task and send it back to thetop of its FOREVER loop.

If (the program running was a HOLD program)

Send a message to the printer task to print the hold information.

Write a status record to the history file.

Set {normal₋₋ power} flag so PID will use the normal power equation.

Return the user interface to its idle state.

Display an abort message.

End of Forever Loop

Display Task Overview

The purpose of the real time display task is to display temperatures,timers, sensor readings, ADC channel readings, and other parameters thatneed to be continually updated every half second.

Display Task Pseudocode:

Initialize display task variables.

Do Forever

Wait for a message every half second from the timer task.

Go to sleep until the message is received.

When awakened, check if another task has sent a list of parameters todisplay or a flag to halt the current update.

Toggle the half second flag {half₋₋ sec}.

If (there's a list of parameters to display) then

Set a semaphore so no one else will update the display.

Turn off the cursor.

While (stepping through the list of parameters)

If (it is a time parameter) then

Display the time.

If (half second flag {half₋₋ sec}is set) then

Increment or decrement the time variable.

Else if (it is a decimal number) then

Display a decimal number.

Else if (it is an integer number) then

Display the integer.

Else if (it is an ADC channel readout) then

Read the counts from the ADC channel.

If (need it displayed as mV) then

Convert counts to mV.

Display the value.

Else if (it is a power display) then

Display the power in terms of watts.

Else if (it is the hours left parameter) then

Convert seconds to tenths of hours.

Display the hours left in tenths of hours.

If (half second flag {half₁₃ sec}is set) then

Decrement the seconds variable.

If (the cursor was on) then

Turn it back on.

Store the current system time in battery RAM.

Clear the semaphore to release the display.

End of Forever Loop

Printer Task Overview

The purpose of the printer task is to handle the runtime printing. It isa low priority task and should not interfere with other time criticaltasks.

Printer Task Pseudocode:

Do Forever

Wait for a message from another task that wishes to print.

Go to sleep until a message is received.

When awaken, make local copies of the global variables to be printed.

Post a printer acknowledgement message.

If (need to print a status or error message) then

Print the information contained in the current history record.

Else if (need to print the page header) then

Print the company name, instrument ID, firmware version number and thecurrent system time and date.

Else if (need to print the program header) then

Print the type of program and its number.

Else if (need to print the program configuration parameters) then

Print the tube type, reaction volume and the sample temperaturedeviation from setpoint that starts the clock.

Else if (need to print end of cycle information) then

Print the ending time and temperature.

Else if (need to print segment information) then

Print either the ramp or hold segment information.

Else if (need to print a pause status message) then

Print the amount of time paused for and at what temp.

End of Forever Loop

LED Task Overview

The purpose of the LED task is to make the illumination of the "Heating"LED reflect the power applied to the main heater. This is a low prioritytask that runs once a second.

LED Task Pseudocode:

Initialize LED task variables.

Do Forever

Send a message to the system to wake this task every second.

Go to sleep.

When awaken, load counter 2 of PIC timer A with a value that reflectsthe power applied to the main heater as follows:

load counter with value={K₋₋ htled}* {ht₋₋ led}

Where:

{K₋₋ htled}holds a constant to compute the time to pulse the heating LEDand is equal to 15200 / 500. 15200 is a little greater than the PIC'sclock of 14.4KHz and this is the value loaded into the timer to keep theLED constantly on. 500 is the main heater power.

{ht₋₋ led}will be a value between 0 and 500 and will be equal to thewatts applied to the main heater.

End of Forever Loop

Link Task Overview

The purpose of the link task is to simulate the user pressing the STARTkey on the keypad. This task is necessary so that programs can beexecuted one right after the other (as in a method) without userintervention. The link task wakes up the sequence task and it beginsrunning the next program as if the START key were pressed.

Link Task Pseudocode:

Initialize link task variables.

Do Forever

If (the flag {weird₋₋ flag}is set and it is not the first file in

the method) then

Send a message to the sequence task to wake up and run a program.

End of Forever Loop

Start Up Sequence POWER-UP SEQUENCE

When the power to the instrument is turned on or the software does aRESET, the following sequence takes place. Note: the numbers belowcorrespond to numbers on the flow chart in FIG. 55.

1. Transmit a Ctrl-G (decimal 7) character out the RS-232 printer port.Poll the RS-232 port for at least 1 second and if a Ctrl-G is received,it is assumed that an external computer is attached to the port and allcommunication during the power-up sequence will be redirected from thekeypad to the RS-232 port. If no Ctrl-G is received, the power-upsequence continues as normal.

2. Check if the MORE key is depressed. If so, go straight to theservice-only hardware diagnostics.

3. The next 3 tests are an audio/visual check and cannot report anerror: 1) the beeper beeps 2) the hot, cooling, and heating LEDs on thekeypad are flashed 3) each pixel of the display is highlighted. Thecopyright and instrument ID screens are displayed as the power-updiagnostics execute.

4. Should an error occur in one of the power-up diagnostics, the name ofthe component that failed is displayed and the keypad is locked to thecustomer. The code `MORE 999` gains access to the service-only hardwarediagnostics.

5. Check channel 0 of the PPI-B device to see if the automated test bitis pulled low. If it is, run the UART test. If the test passes, beep thebeeper continuously.

6. Start the CRETIN operating system which in turn will start up eachtask by priority level.

7. Check a flag in battery RAM to see if the instrument has beencalibrated. If not, display an error message and lock the keypad to thecustomer. The code `MORE 999` gains access to the service-onlycalibration tests.

8. Run a test that measures the voltage and line frequency and see ifboth these values match the configuration plug selected whilecalibrating the instrument. If not, display an error message and lockthe keypad to the customer. The code `MORE 999` gains access to theservice-only calibration tests.

9. Check a flag in battery RAM to see if the instrument has beeninstalled. If not, display an error message and lock the keypad to thecustomer. The code `MORE 999` gains access to the install routine.

10. Check a flag in battery RAM to see if there was a power failurewhile the instrument was running. If so, start a 4° C. soak and displaythe amount of time the power was off for. Ask the user if they wish toview the history file which will tell them exactly how far along theywere in the run when the power went off. If they select yes, they gostraight to the user diagnostics.

11. Beep the beeper.

12. Display the top level user interface screen.

There now follows information concerning two sets of user interfacescreens, the first of which is produced by Version 1 of the Electronicsand Software and the second of which is produced by Version 2 of theElectronics and Software. ##SPC1##

What we claim is:
 1. An apparatus for controlled automated performanceof polymerase chain reactions in at least one sample tube containing aknown volume of a liquid sample mixture, which apparatus comprises:a. Asample block having at least one well for said at least one sample tube,b. a computing apparatus, c. heating and cooling means controlled bysaid computing apparatus for changing the temperature of said sampleblock, and d. means for determining the temperature of said block in afirst sample interval, wherein said first sample interval is an intervalof time designated as time n;wherein said computing apparatus includesmeans for determining the temperature of said liquid sample mixture as afunction of the temperature of said sample block over time by utilizingthe relationship:

    Tsamp.sub.n =Tsamp.sub.n-1 +TB.sub.n -Tsamp.sub.n-1) * tinterval/tau

where Tsamp_(n) is equal to the sample temperature in said first sampleinterval, Tsamp_(n-1) is a sample temperature in a second sampleinterval immediately preceding the first sample interval, said secondsample interval designated as time n-1 , TB_(n) is equal to the blocktermperature in said first sample interval, tinterval is a time inseconds between consecutive sample intervals, and tau is a function ofthermal characteristics of said apparatus.
 2. The apparatus of claim 1,wherein said thermal characteristics comprise a first thermal timeconstant corresponding to said at least one sample tube and said volumeof said sample mixture, and a second thermal time constant correspondingto a block temperature sensor thermally coupled to said block.
 3. Theapparatus of claim 2, wherein tau equals approximately said firstthermal time constant minus said second thermal time constant.
 4. Theapparatus of claim 3, wherein said first thermal time constant isbetween approximately 5 seconds and 14 seconds and said second thermaltime constant equals approximately 1.5 seconds.
 5. The apparatus ofclaim 1 further comprising an input device for receiving one or moreuser defined setpoints, each said setpoint defining a hold time / firsttemperature pair; and wherein said computing apparatus includes meansfor controlling said heating and cooling means as a function of saiduser defined setpoints and said sample temperature; and wherein saidcomputing apparatus further comprises means to combine one or moresetpoints in a user defined order to form a profile wherein thecontrolling of said heating and cooling means as a function of thesetpoints in a said profile defines a profile run.
 6. The apparatus ofclaim 7, wherein said first temperature defines a target sampletemperature after ramping said sample temperature at a preselected ramprate, and said hold time defines a length of time to maintain saidsample at said first temperature after ramping.
 7. The apparatus ofclaim 1 further including an input device for receiving user definedsetpoints defining a hold time/temperature profile, wherein saidcomputing apparatus includes means for controlling said heating andcooling means as a function of said user defined setpoints and saidsample temperature.
 8. The apparatus of claim 6, wherein said apparatusfor controlled automated performance of polymerase chain reactionsincludes an enclosure for said sample block defining an enclosed ambientatmosphere, and wherein said sample block is comprised of a centralregion containing in an upper surface an array of sample wells forholding said at least one sample tube, an end edge region comprising twoend edges at opposite ends of said block which are in thermal contactwith said enclosed ambient atmosphere, and a manifold region comprisingtwo manifold edges at opposite sides of said block, wherein each saidmanifold edge is thermally coupled to a manifold.
 9. The apparatus ofclaim 8 wherein said heating means is a heater having a central heatingzone thermally coupled to said central region, an end edge heating zonethermally coupled to said edge region, and a manifold heating zonethermally coupled to said manifold region.
 10. The apparatus of claim 8wherein said computing apparatus comprises means for determining anactual heating power to be applied to each said heating zone in saidfirst sample interval, including:a. means for determining a theoreticalsecond power representing the total power to apply to said block in saidsample interval without accounting for power losses, b. means fordetermining theoretical third powers to be applied to each said zone insaid first sample interval as a function of said theoretical secondpower, c. means for determining power losses by said regions in saidfirst sample interval, and d. means for determining the actual heatingpower to be applied to each said heating zone as a function of saidtheoretical third powers and said power losses by said regions.
 11. Theapparatus of claim 10 wherein said means for determining saidtheoretical second power includes:a. means for determining a firstfraction of the difference between the target sample temperature afterramping and the sample temperature in said second sample interval to bemade up in said first sample interval, and b. means for determining saidtheoretical second power as a function of said first fraction, saidsample temperature in said second sample interval and said blocktemperature in said second sample interval.
 12. The apparatus of claim11 wherein said means for determining said theoretical second powerincludes means for determining said theoretical second power as afunction of:

    CP * P/t.sub.interval * ((SP-T.sub.samp.sbsb.n-1) * F * tau/t.sub.interval +T.sub.samp.sbsb.n-1 -T.sub.B.sbsb.n-1)

where CP is equala to a thermal mass of said block P is a proportionalterm gain, SP is said target sample temperature after ramping, F is saidfirst fraction and T_(B).sbsb.n-1 is equal to the temperature of theblock in said second sample interval.
 13. The apparatus of claim 10wherein said computing apparatus comprises means for adjusting saidtheoretical second power when said sample temperature in said secondsample interval is within an integral band of said target sampletemperature after ramping, in order to close out remaining error. 14.The apparatus of claim 13, wherein said integral band is approximately ⁺/₋ 0.5 ° C.
 15. The apparatus of claim 13 wherein said means foradjusting said theoretical second power comprises means for adjustingsaid theoretical second power as a function of a power adjustment termgiven approximately by:

    int.sub.-- sum.sub.n =int.sub.-- sum.sub.1-1 +(SP -T.sub.samp.sbsb.n-1)

    pwr.sub.-- adj=ki * int.sub.-- sum.sub.n

where pwr₋₋ adj equals said power adjustment term, int₋₋ sum_(n) is avalue of an accumulating integral term in said first sample interval,int₋₋ sum_(n-1) is a value of said accumulating integral term in saidsecond sample interval, SP is said target sample temperature afterramping, and ki equals an integral gain.
 16. The apparatus of claim 15,wherein said integral gain is approximately
 512. 17. The apparatus ofclaim 10, wherein said means for determining power losses by saidregions in said first sample interval comprises:a. means for determiningpower lost to a foam backing on said sample block in said first sampleinterval, b. means for determining power lost to said manifolds in saidfirst sample interval, and c. means for determining power lost to saidenclosed ambient atmosphere in said first sample interval.
 18. Theapparatus of claim 17, wherein said means for determining power lost tosaid foam backing on said sample block in said first sample intervalcomprises:a. means for determining the temperature of said foam backingin said first sample interval, b. means for determining said power lostto said foam backing as a function of said temperature of said foambacking in said first sample interval, said temperature of said block insaid first sample interval, and a thermal mass of said foam backing. 19.The apparatus of claim 18, wherein said means for determining thetemperature of the foam backing in said first sample interval comprisesmeans for determining the temperature of the foam backing in said firstsample interval according approximately to:

    T.sub.foam.sbsb.n =T.sub.foam.sbsb.n-1 +(T.sub.B.sbsb.n -T.sub.foam.sbsb.n-1) * t.sub.interval / tau2

where T_(foam).sbsb.n is equal to the temperature of the foam backing insaid first sample interval, T_(foam).sbsb.n-1 is equal to the foambacking temperature in said second sample interval, and tau2 is afunction of said thermal mass of said foam backing.
 20. The apparatus ofclaim 19, wherein tau2 is approximately
 30. 21. The apparatus of claim18 wherein said means for determining the power lost to said foambacking comprises means for determining the power lost to said foambacking according approximately to:

    foam-pwr=C * (T.sub.B.sbsb.n -T.sub.foam.sbsb.n)

where foam-pwr is said power lost to said foam backing in said firstsample interval, T_(foam).sbsb.n is equal to the temperature of the foamin said first sample interval and C is equal to the thermal mass of thefoam backing.
 22. The apparatus of claim 17 additionally comprisingmeans for delivering a bias coolant constantly applied to said sampleblock, and wherein said means for determining the power lost to saidmanifolds in said first sample interval comprises means for determiningthe power lost to said manifolds in said first sample interval accordingapproximately to:

    manifold.sub.-- loss=KA (T.sub.B.sbsb.n -T.sub.A.sbsb.n)+KD (T.sub.B.sbsb.n -T.sub.C.sbsb.n)+TM (dT/dt)

where manifold₋₋ loss equals said power lost to said manifolds in saidfirst sample interval, KA equals an end edge region-to-enclosed ambientatmosphere conductance constant, T_(A).sbsb.n equals the enclosedambient atmosphere temperature in said first sample interval,T_(C).sbsb.n equals the temperature of said bias coolant in said firstsample interval, KC equals a sample block-to-coolant conductanceconstant, TM equals a thermal mass of said manifolds and dT/dt equalssaid preselected ramp rate.
 23. The apparatus of claim 17 additionallycomprising means for delivering a bias coolant constantly applied tosaid sample block, and wherein said means for determining the power lostto said enclosed ambient atmosphere in said first sample intervalcomprises means for determining the power lost to said enclosed ambientatmosphere in said first sample interval according approximately to:

    ambient.sub.-- loss=K2A (T.sub.B.sbsb.n -T.sub.A.sbsb.n)+K2C (T.sub.B.sbsb.n -T.sub.C .sbsb.n) +TM2(dT/dt)

where ambient₁₃ loss is said power lost to said enclosed ambientatmosphere in said first sample interval, K2A equals an end edgeregion-to-enclosed ambient atmosphere conductance constant, T_(A).sbsb.nequals the enclosed ambient atmosphere temperature in said first sampleinterval, K2C equals an end edge region-to-coolant conductance constant,T_(C).sbsb.n equals the temperature of the bias coolant in said firstsample interval, TM2 equals a thermal mass of said enclosed ambientatmosphere, and dT/dt equals said preselected ramp rate.
 24. Theapparatus of claim 10, wherein said means for determining the actualheating power to be applied to each said heating zone as a function ofsaid theoretical third powers and said power losses by said regionscomprises means for determining the actual heating power to be appliedto each said heating zone as a function of said theoretical third powersand said power losses by said regions according approximately to:

    central.sub.-- pwr=pwr * cper

ti manifold₋₋ pwr=pwr * mper+manifold₋₋ loss

    edge.sub.-- pwr=pwr * eper+ambient.sub.-- loss

where pwr is a function of said theoretical second power plus power lostto a foam backing on said block in said first sample interval,manifold₋₋ loss equals a power lost to said manifolds in said firstsample interval, ambient₋₋ loss equals a power lost in said edge regionto said enclosed ambient atmosphere in said first sample interval,central₋₋ pwr equals a power to be applied to said central heating zonein said first sample interval, manifold₋₋ pwr equals a power to beapplied to said manifold heating zone in said first sample interval,edge₋₋ pwr equals a power to be applied to said end edge heating zone insaid first sample interval, cper equals a percentage of heater area insaid central heating zone, mper equals a percentage of heater area insaid manifold heating zone, and eper equals a percentage of heater areain said edge heating zone.
 25. The apparatus of claim 24, wherein cperequals approximately 0.66, mper equals approximately 0.20 and eperequals approximately 0.14.
 26. The apparatus of claim 8, wherein saidsample block contains multiple transverse bias cooling channelsalternating with multiple transverse ramp cooling channels, said biasand ramp cooling channels being parallel to said upper surface, saidapparatus further comprising means for constantly pumping chilledcoolant through said bias cooling channels and means for selectivelypumping chilled coolant through said ramp cooling channels comprisingvalve means controlled by said computing apparatus.
 27. The apparatus ofclaim 26 wherein said computing apparatus comprises means fordetermining a total cooling power to be applied to said block in saidfirst sample interval including:a. means for determining a theoreticalcooling power representing the total power to apply to said block insaid first sample interval without accounting for power losses, b. meansfor determining power losses in said block regions in said first sampleinterval, and c. means for determining said total cooling power as afunction of said theoretical cooling power and said power losses. 28.The apparatus of claim 27 wherein said means for selectively pumpingchilled coolant through said ramp cooling including further comprises:a.means for determining that ramp direction is downward, b. means fordetermining a cooling breakpoint as a function of said block temperatureand a temperature of said coolant, and c. means for determining ifcoolant will be pumped through said ramp cooling channels as a functionof said total cooling power and said cooling breakpoint, wherein thedetermination made by said means for determining if coolant will bepumped through said ramp cooling channels constitutes a ramp coolingdecision.
 29. The apparatus of claim 27 wherein said means fordetermining said theoretical cooling power includes:a. means fordetermining a second fraction of the difference between the targetsample temperature after ramping and the sample temperature in saidsecond sample interval to be made up in said first sample interval, andb. means for determining said theoretical cooling power as a function ofsaid second fraction, said sample temperature in said second sampleinterval and said block temperature in said second sample interval. 30.The apparatus of claim 29, wherein said means for determining saidtheoretical cooling power comprises means for determining saidtheoretical cooling power as a function of:

    CP * P/t.sub.interval * ((SP-T.sub.samp.sbsb.n-) * F * tau/t.sub.interval +T.sub.samp.sbsb.n-1 -T.sub.B.sbsb.n-1

where CP is equal to a thermal mass of said block, P is a proportionalterm gain, SP equals said target sample temperature after ramping,T_(B).sbsb.n-1 is equal to the temperature of the block in said secondsample interval, F is said second fraction and T_(B).sbsb.n-1 is equalto the temperature of the block in said second sample interval.
 31. Theapparatus of claim 27 wherein said computing apparatus comprises meansfor adjusting said theoretical cooling power when said sampletemperature in said second sample interval is within an integral band ofsaid target sample temperature after ramping, in order to close outremaining error.
 32. The apparatus of claim 31, wherein said integralband is approximately ⁺ /₃₁ 0.5° C.
 33. The apparatus of claim 31,wherein said means for adjusting said theoretical cooling powercomprises means for adjusting said theoretical cooling power as afunction of a power adjustment term given approximately by:int₋₋ sum_(n)=int₋₋ sum_(n-1) +(SP-T_(samp) _(n-1))

    pwr.sub.-- adj=ki * int.sub.-- sum.sub.n

where pwr₋₋ adj equals said power adjustment term, int₋₋ sum_(n) is avalue of an accumulating integral term in said first sample interval,int₋₋ sum_(n-1) is a value of said accumulating integral term in saidsecond sample interval, SP equals said target sample temperature afterramping, and ki equals an integral gain.
 34. The apparatus of claim 33,wherein said integral gain is approximately
 512. 35. The apparatus ofclaim 27, wherein said means for determining power losses in said blockregions in said first sample interval comprises:a. means for determiningpower lost to a foam backing on said sample block in said first sampleinterval b. means for determining power lost to said manifolds in saidfirst sample interval, and c. means for determining power lost to saidenclosed ambient atmosphere in said first sample interval.
 36. Theapparatus of claim 35, wherein said means for determining power lost tosaid foam backing on said sample block in said first sample intervalcomprises:a. means for determining the temperature of said foam backingin said first sample interval, b. means for determining said power lostto said foam backing as a function of said temperature of said foambacking in said sample interval, said temperature of said block in saidfirst sample interval, and a thermal mass of said foam backing.
 37. Theapparatus of claim 36, wherein said means for determining thetemperature of the foam backing in said first sample interval comprisesmeans for determining the temperature of the foam backing in said firstsample interval according approximately to:

    T.sub.foam.sbsb.n =T.sub.foam.sbsb.n-1 30 (T.sub.B.sbsb.n -T.sub.foam.sbsb.n-1) * t.sub.interval / tau2

where T_(foam).sbsb.n is equal to the temperature of the foam backing insaid first sample interval, T_(foam).sbsb.n-1 is equal to the foambacking temperature in said second sample interval, and tau2 is afunction of said thermal mass of said foam backing.
 38. The apparatus ofclaim 37, wherein tau2 is approximately
 30. 39. The apparatus of claim37, wherein said means for determining the power lost to said foambacking comprises means for determining the power lost to said foambacking according approximately to:foam-pwr=C * (T_(B).sbsb.n-T_(foam).sbsb.n) where foam-pwr is said power lost to said foam backingin said first sample interval, T_(foam).sbsb.n is equal to thetemperature of the foam in said first sample interval and C is equal tothe thermal mass of the foam backing.
 40. The apparatus of claim 35,wherein said means for determining the power lost to said manifolds insaid first sample interval comprises means for determining the powerlost to said manifolds in said first sample interval accordingapproximately to:

    manifold.sub.-- loss=KA (T.sub.B.sbsb.n -T.sub.A.sbsb.n)+KC (T.sub.B.sbsb.n -T.sub.C.sbsb.n) +TM (dT/dt)

where manifold₋₋ loss equals said power lost to said manifolds in saidfirst sample interval, KA equals an end edge region-to-enclosed ambientatmosphere conductance constant, T_(A).sbsb.n equals the enclosedambient atmosphere temperature in said first sample interval,T_(C).sbsb.n equals a temperature of said bias coolant in said firstsample interval, KC equals a sample block-to-coolant conductanceconstant, TM equals a thermal mass of said manifolds and dT/dt equalssaid preselected ramp rate.
 41. The apparatus of claim 35, wherein saidmeans for determining the power lost to said enclosed ambient atmospherein said first sample interval comprises means for determining the powerlost to said enclosed ambient atmosphere in said first sample intervalaccording approximately to:

    ambient.sub.-- loss=K2A (T.sub.B.sbsb.n -T.sub.A.sbsb.n)+K2C (T.sub.B.sbsb.n -T.sub.C.sbsb.n)+TM2(dT/dt)

where ambient₋₋ loss is said power lost to said enclosed ambientatmosphere in said first sample interval, K2A equals an end edgeregion-to-enclosed ambient atmosphere conductance constant, T_(A).sbsb.nequals the enclosed ambient atmosphere temperature in said first sampleinterval, K2C equals an end edge region-to-coolant constant,T_(C).sbsb.n equals the coolant temperature in said first sampleinterval, TM2 equals a thermal mass of said enclosed ambient atmosphere,and dT/dt equals said preselected ramp rate.
 42. The apparatus of claim28, wherein said cooling breakpoint is a function of the differencebetween said block temperature in said first sample interval and saidtemperature of said coolant fluid in said first sample interval.
 43. Theapparatus of claim 28, wherein said means for determining if said rampcooling channels will be open further comprises means for determining ifsaid ramp cooling channels will be open as a function of the differencebetween said total cooling power and said cooling breakpoint.
 44. Theapparatus of claim 1, further comprising means for overshooting thetemperature of said sample block above a desired sample temperature,thereby decreasing an upramp time required for said liquid samplemixture to achieve said desired sample temperature.
 45. The apparatus ofclaim 44 further comprising means for controlling the overshoot suchthat it is equal to or less than approximately 0.5° C.
 46. The apparatusof claim 15 additionally comprising means for delivering a bias coolantconstantly applied to said sample block, and wherein said means foradjusting said theoretical second power comprises means for dynamicallymodifying said integral gain when said sample temperature is within saidintegral band of said target sample temperature, and said apparatus isperforming an upramp, according approximately to:

    Gain.sub.n= C1*(SP-TC.sub.n, +C2

Where Gain_(n) is said integral gain in said first sample interval, C1is a first gain constant, SP is said target sample temperature afterramping, TC_(n) is the temperature of said coolant in said first sampleinterval and C2 is a second gain constant.
 47. The apparatus of claim46, wherein said Cl equals approximately 0.65.
 48. The apparatus ofclaim 46, wherein said C2 equals approximately
 109. 49. The apparatus ofclaim 46, wherein said C2 equals approximately
 105. 50. The apparatus ofclaim 46 wherein a gain constant is used for said integral gain whensaid sample temperature is within a second integral band of said targetsample temperature, said second integral band being smaller than saidintegral band.
 51. The apparatus of claim 50, wherein said integral bandis approximately ±11° C. and said second integral band is approximately±0.5° C.
 52. The apparatus of claim 50, wherein said gain constant isapproximately
 512. 53. The apparatus of claim 1, further comprisingmeans for undershooting the temperature of said sample block below adesired sample temperature, thereby decreasing a downramp time requiredfor said liquid sample mixture to achieve said desired sampletemperature.
 54. The apparatus of claim 53 further comprising means forcontrolling the undershoot such that it is equal to or less thanapproximately 0.5° C.
 55. The apparatus of claim 28 further comprisingmeans for dynamically changing said means for determining saidtheoretical cooling power such that the temperature of the block willundershoot said target sample temperature after ramping in a controlledfashion.
 56. The apparatus of claim 55 further comprising means foractivating, as a function of said target sample temperature afterramping, ramp direction, and said ramp cooling decision, said means fordynamically changing said means for determining said theoretical coolingpower.
 57. The apparatus of claim 54, wherein said means for activatingsaid means for dynamically changing said means for determining saidtheoretical cooling power comprises means for activating said means fordynamically changing said means for determining said theoretical coolingpower if said target sample temperature after ramping is greater thanapproximately 45° C., ramp direction is downward and said ramp coolingdecision is not to ramp.
 58. The apparatus of claim 55, wherein saidmeans for dynamically changing said means for determining saidtheoretical cooling power further comprises means for determining saidtheoretical cooling power as a function of:

    CP P/t.sub.interval *((SP-T.sub.B.sbsb.n-1)*F*tau/t.sub.interval)


59. The apparatus of claim 1, wherein said means for determining thetemperature of said block in said first sample interval comprises meansfor determining the temperature of said block in said first sampleinterval as a function of at least one temperature of said block in aprevious sample interval.
 60. The apparatus of claim 1, wherein saidmeans for determining the temperature of said block in said first sampleinterval comprises means for reading the temperature of said block froma block temperature sensor thermally coupled to said block.
 61. Theapparatus of claim 1, wherein t_(interval) equals approximately 0.2seconds.
 62. The apparatus of claim 1, wherein said known volume ofliquid sample mixture is in the range of approximately 20-100microliters.
 63. The apparatus of claim 5, wherein said computingapparatus comprises means for receiving user issued commands to invokesaid profile runs, wherein said invoking causes said profiles to be runat least once.
 64. The apparatus of claim 63, wherein said input devicefurther comprises means for receiving a user defined cycle count numberfor a plurality of said profiles, and wherein said computing meanscomprises means for running said profiles said cycle count number oftimes automatically when said profiles are invoked.
 65. The apparatus ofclaim 64, wherein said computing apparatus further comprises means forlinking multiple profiles in any order to form a protocol, said protocoldefining a sequence of said profiles to be invoked automatically uponsaid command from the user, wherein invoking said sequence of profilesautomatically defines a protocol run.
 66. The apparatus of claim 65,wherein said computing apparatus further comprises means for linking asingle profile a plurality of times in a single protocol.
 67. Theapparatus of claim 65, wherein said computing apparatus furthercomprises means for storing a plurality of protocols.
 68. The apparatusof claim 67, wherein said computing apparatus comprises means forincluding any said profile in a plurality of said protocols.
 69. Theapparatus of claim 65, wherein said computing apparatus comprises meansfor protecting a profile included in any said protocol from beingdeleted or overwritten.
 70. The apparatus of claim 5, further comprisingmeans for determining that an electrical power to operate said apparatuswent off during a said profile run.
 71. The apparatus of claim 70further comprising means for reporting the length of said electricalpower outage when said electrical power is restored.
 72. The apparatusof claim 70, further comprising means for automatically starting a soakof said liquid sample mixture upon restoration of said electrical power.73. The apparatus of claim 72, wherein said soak occurs at approximately4° C.
 74. The apparatus of claim 64, further comprising means forautomatically increasing the hold time of any or all said setpoints fromcycle to cycle in said cycle count.
 75. The apparatus of claim 74,wherein activation of said means for automatically increasing the holdtime of any or all setpoints from cycle to cycle is selectable as a userlevel option.
 76. The apparatus of claim 74, wherein said computingapparatus comprises means for receiving first user defined valuesdefining an increment in hold time from cycle to cycle, and wherein saidmeans for automatically increasing the hold time comprises means forautomatically increasing the hold time of any or all said setpoints fromcycle to cycle as a first function of said first user defined values.77. The apparatus of claim 76, wherein said first function is a linearfunction.
 78. The apparatus of claim 76, wherein said first function isa geometric function.
 79. The apparatus of claim 64 further comprisingmeans for automatically decreasing the hold time of any or all saidsetpoints from cycle to cycle in said cycle count.
 80. The apparatus ofclaim 79, wherein activation of said means for automatically decreasingthe hold time of any or all setpoints from cycle to cycle is selectableas a user level option via said input device.
 81. The apparatus of claim79, wherein said computing apparatus comprises means for receivingsecond user defined values defining a decrement in hold time from cycleto cycle, and wherein said means for automatically decreasing the holdtime comprises means for automatically decreasing the hold time of anyor all said setpoints from cycle to cycle as a second function of saidsecond user defined values.
 82. The apparatus of claim 81, wherein saidsecond function is a linear function.
 83. The apparatus of claim 81,wherein said second function is a geometric function.
 84. The apparatusof claim 64 further comprising means for automatically increasing thefirst temperature of any or all said setpoints from cycle to cycle insaid cycle count.
 85. The apparatus of claim 84, wherein activation ofsaid means for automatically increasing the first temperature of any orall setpoints from cycle to cycle is selectable as a user level option.86. The apparatus of claim 84, wherein said computing apparatuscomprises means for receiving third user defined values defining anincrement in the first temperature from cycle to cycle, and wherein saidmeans for automatically increasing the first temperature comprises meansfor automatically increasing the first temperature of any or allsetpoints from cycle to cycle as a third function of said third userdefined values.
 87. The apparatus of claim 86, wherein said thirdfunction is a linear function.
 88. The apparatus of claim 86, whereinsaid third function is a geometric function.
 89. The apparatus of claim64 further comprising means for automatically decreasing the firsttemperature of any or all said setpoints from cycle to cycle in saidcycle count.
 90. The apparatus of claim 89, wherein activation of saidmeans for automatically decreasing the first temperature of any or allsetpoints from cycle to cycle is selectable as a user level option. 91.The apparatus of claim 89, wherein said computing apparatus comprisesmeans for receiving fourth user defined values defining a decrement infirst temperature from cycle to cycle, and wherein said means forautomatically decreasing the first temperature comprises means forautomatically decreasing the first temperature of any or all setpointsfrom cycle to cycle as a fourth function of said fourth user definedvalues.
 92. The apparatus of claim 91, wherein said fourth function is alinear function.
 93. The apparatus of claim 91, wherein said fourthfunction is a geometric function.
 94. The apparatus of claim 65, furthercomprising a programmed pause option means to automatically suspend asaid profile run for a user defined period of time.
 95. The apparatus ofclaim 94, further comprising a programmed pause option means toautomatically suspend a said protocol run for a user defined period oftime.
 96. The apparatus of claim 95, wherein said pause option meanscomprises means to suspend a said profile run at any point in a saidprofile run or during any or all cycles of a said profile invocation,and further comprising means for suspending a said protocol run afterany or all said profile runs in a said protocol run.
 97. The apparatusof claim 6 further comprising user-controllable means for defining atemperature range such that said computing apparatus will commencetiming said hold time when said sample temperature is within saidtemperature range of said target sample temperature.
 98. The apparatusof claim 2, further comprising an input device for receiving a tube typeand a reaction volume, and wherein said computing apparatus comprisesmeans for determining said thermal time constant for said sample tube asa function of said tube type and said sample volume.
 99. The apparatusof claim 9, further comprising means for performing diagnostic checks ofsaid heating means.
 100. The apparatus of claim 99, wherein saiddiagnostic checks of said heating means comprises one or more heaterping tests, block thermal capacity tests, ramp cooling conductancetests, sensor lag tests.
 101. The apparatus of claim 99, furthercomprising means for performing diagnostic checks of a cooling capacityof said apparatus.
 102. The apparatus of claim 101, wherein saiddiagnostic checks comprise one or more control cooling conductancetests, block thermal capacity tests, chiller tests, ramp coolingconductance tests, sensor lag tests, coolant capacity tests.
 103. Theapparatus of claim 1, further comprising means for performing hardwarediagnostics on user demand and/or automatically upon system start-up.104. The apparatus of claim 103, wherein said hardware diagnosticsinclude tests of one or more of a Programmable Peripheral Interfacedevice, Battery RAM device, Battery RAM checksum, EPROM devices,Programmable Interface Timer devices, Clock/Calendar device,Programmable Interrupt Controller device, Analog to Digital Section,RS-232 Section, Display Section, Keyboard, Beeper, Ramp Cooling Values,EPROM mismatch, Firmware version level, Battery RAM Checksum andInitialization, Autostart Program Flag, Clear Calibration Flag, HeatedCover heater and control circuitry, Edge heater and control circuitry,Manifold heater and control circuitry, Central heater and controlcircuitry, Sample block thermal cutoff, Heated cover thermal cutoff.105. The apparatus of claim 60 further comprising means for adjustingtemperature sensor readings from said block temperature sensor toaccount for drift in analog circuitry.
 106. The apparatus of claim 105wherein said means for adjusting temperature sensor readings from saidblock temperature sensor to account for drift in the analog circuitryfurther comprises means for determining said drift, wherein said meansfor determining are capable of:a) Measuring one or more test voltagesunder controlled conditions, b) Reading said voltages at the start ofeach run, and c) Measuring said drift as a function of said testvoltages under controlled conditions and said voltages at the start ofeach run.
 107. The apparatus of claim 65, wherein said computingapparatus comprises a means to display the approximate amount of timeleft in a said profile run or in a said protocol run.
 108. The apparatusof claim 65, wherein said computing apparatus comprises means fordisplaying, during a said profile or protocol run, the sampletemperature at any given time in said profile or protocol run.
 109. Theapparatus of claim 6, further comprising means for determining, for oneor more setpoints, a first difference between said sample temperatureafter said hold time has expired and said target sample temperature ofsaid setpoint.
 110. The apparatus of claim 109, further comprising meansfor receiving and storing a user defined temperature differential. 111.The apparatus of claim 110, wherein said computing apparatus comprisesmeans to report an error if said user defined temperature differentialis greater than said first difference.
 112. The apparatus of claim 6,further comprising means for configuring a second temperature, andfurther comprising means for causing said heating and cooling means tomaintain said sample at said second temperature during any idle state.113. The apparatus of claim 6, further comprising means for determiningthat, for one or more setpoints, said first temperature is reachedwithin a user definable amount of time.
 114. The apparatus of claim 89,further comprising means for determining whether said automaticallydecreased first temperature remains within a defined range.
 115. Theapparatus of claim 84, further comprising means for determining whethersaid automatically increased first temperature remains within a definedrange.
 116. The apparatus of claim 79, further comprising a means todetermine whether said automatically decreased hold time is negative.117. The apparatus of claim 60 further comprising means to continuallymonitor said block sensor and to invoke an abort procedure if blocksensor readings are above a maximum desireable temperature for saidblock by a predetermined number of degrees for a predetermined number oftimes.
 118. The apparatus of claim 117, wherein said abort procedurecomprises means for one or more of aborting the profile or protocol run,flagging an error in a history file, displaying message alerts to auser, disabling said heating means.
 119. The apparatus of claim 65,further comprising means for printing information stored in said system.120. The apparatus of claim 119, wherein said information comprises oneor more of: contents of a profile, contents of a protocol, listing ofcreated profiles, listing of created protocols, configurationparameters, system calibration parameters.
 121. The apparatus of claim1, further comprising means for controlling all user controllablefunctions remotely.
 122. The apparatus of claim 1 wherein said computingapparatus comprises means to display a menu driven user interface. 123.The apparatus of claim 65 wherein said computing apparatus maintains ahistory file of an immediately previous run containing details of saidprevious run suitable for integrity checks and error analysis. 124.Thermocycler apparatus for automated performance of a polymerase chainreaction comprisinga. a metal sample block having a major top surfaceand a major bottom surface, b. an array of spaced-apart sample wellsformed in said major top surface, c. bias cooling constantly applied tosaid sample block at a rate sufficient to cause said block, if at atemperature within the range of 35°-100° C., to cool uniformly at a rateof at least about 0.1° C./sec unless external heat is supplied, and d.computer-controllable heating means for uniformly raising thetemperature of said block at a rate greater than the bias cooling rate,said thermocycler apparatus comprising means for maintaining the arrayof sample wells at a constant in the range of 35°-100° C. within atolerance band of plus or minus about 0.5° C.
 125. Thermocyclerapparatus according to claim 124 wherein said array comprises arectangular array having rows of spaced-apart sample wells. 126.Thermocycler apparatus according to claim 125 wherein said arraycomprises an 8-by-12 rectangular array having center-to-center samplewell spacing compatible with industry standard microliter plate format.127. Thermocycler apparatus according to claim 126 wherein said sampleblock has a block thermal capacity of about 500-600 watt-seconds per °C.128. Thermocycler apparatus according to claim 125 wherein said sampleblock contains multiple transverse bias cooling channels through saidblock parallel to said top surface and parallel to and spaced from therows of wells, and wherein said bias cooling is applied by pumpingcooling liquid through said bias cooling channels.
 129. Thermocyclerapparatus according to claim 128 wherein said bias cooling channels areinsulated.
 130. Thermocycler apparatus according to claim 124 whereinsaid computer-controllable heating means comprises multiple, separatelycontrollable heating zones for said block, at least one first zone forthe portion of the block containing the array of sample wells and atleast one second zone for the peripheral portion of the block outsidethe array.
 131. Thermocycler apparatus according to claim 130 whereinsaid computer-controllable heating means comprises a multizone filmheater in thermal contact with said major bottom surface. 132.Thermocycler apparatus according to claim 124 wherein said sample blockincludes around its periphery a guard band having thermalcharacteristics similar to the block portion containing the array andwherein said guard band is bias cooled and controllably heated. 133.Thermocycler apparatus according to claim 132 wherein said guard bandincludes a groove formed in said top surface extending substantiallyaround said array, decreasing the thermal conductivity between the blockportion containing the array and the guard band.
 134. Thermocyclerapparatus according to claim 132 wherein said computer-controllableheating means comprises multiple, separately controllable heating zonesfor said block, at least one first zone for the portion of the blockcontaining the array of sample wells and at least one second zone forthe guard band.
 135. Thermocycler apparatus according to claim 134wherein said computer-controllable heating means comprises a multizonefilm heater in thermal contact with said major bottom surface. 136.Thermocycler apparatus according to claim 124 further comprisingcomputer-controllable ramp cooling means capable of lowering thetemperature of said block at a rate of at least about 4° C. per secondfrom 100° C. and at least about 2° C. per second from 40° C. 137.Thermocycler apparatus according to claim 136 wherein said arraycomprises a rectangular array comprising rows of spaced-apart samplewells, wherein said sample block contains multiple transverse biascooling channels alternating with multiple transverse ramp coolingchannels, and wherein said bias cooling and said ramp cooling areapplied by pumping cooling liquid through said ramp cooling channels andsaid bias cooling channels.
 138. Thermocycler apparatus according toclaim 137 further comprising means to deliver cooling liquid to oppositeends of successive ramp cooling channels.
 139. Thermocycler apparatusaccording to claim 136 wherein said computer-controllable heating meansis capable of ramp heating.
 140. Thermocycler apparatus according toclaim 139 wherein said controllable heating comprises multiple,separately controllable heating zones for said block, at least one firstzone for the portion of the block containing the array of sample wellsand at least one second zone for the portion of the block outside thearray.
 141. Thermocycler apparatus according to claim 140 wherein saidcomputer-controllable heating means comprises a multizone film heater inthermal contact with said major bottom surface.
 142. Thermocyclerapparatus according to claim 124 further comprising means for seatinginto the wells in said array sample tubes of nonidentical height with aseating force on each sample tube sufficient to cause a snug, flush fitbetween the surface of the tube and the surface of the well. 143.Thermocycler apparatus according to claim 142 wherein said means forseating comprises deformable, compliant, gas-tight caps for said sampletubes, a vertically displaceable platen, and controllable means forforcibly lowering said platen to maintain said seating force on the capfor each tube.
 144. Thermocycler apparatus-according to claim 143wherein said platen is maintained at a heated temperature in the rangeof 94°-110° C.
 145. Thermocycler apparatus according to claim 144wherein said platen is maintained at a temperature in the range of100°-110° C.
 146. Thermocycler apparatus according to claim 124 furthercomprising a computer system for controlling said heating means. 147.Thermocycler apparatus according to claim 136, wherein said computersystem controls said ramp cooling means.
 148. Thermocycler apparatus forautomated, rapid performance of a polymerase chain reaction comprisinga.a thermally homogeneous metal sample block of low thermal mass having amajor top surface and a major bottom surface, said block containing in acentral region of its upper surface an 8-by-12 rectangular array ofsample wells having center-to-center spacing compatible with industrystandard microtiter plate format, said block also containing aperipheral region surrounding said array, said peripheral regioncomprising a guard band having thermal characteristics similar to thethermal characteristics of the central region, b. a bias cooling systemfor constantly cooling said sample block at a bias cooling ratesufficient to cause said block, if at a temperature within the range of35°-100° C., to cool uniformly at a rate of at least about 0.1° C./secunless external heat is supplied, c. a computer system for receiving andstoring user data regarding times and temperatures defining a pluralityof reaction cycles, d. a ramp cooling system controlled by said computersystem for selectively cooling said sample block at a ramp cooling rateof at least about 4° C./sec from 100° C. and at least about 2° C./secfrom 40° C., e. a multizone heating system controlled by said computersystem having a heating zone for the central region of the block and aheating zone for the guard band, said heating system comprising meansfor providing heat necessary to maintain the sample block at a constanttemperature in the range of 35°-100° C. and also capable of providingramp heating to the block, f. a pressing cover vertically displaceableabove said sample block, and g. cover displacing means for raising saidcover and for lowering said cover and maintaining its vertical positionagainst a resisting force of at least about 3000 grams, saidthermocycler apparatus comprising means for maintaining the array ofsample wells at a constant temperature in the range of 35°-100° C.within a tolerance band of plus or minus 0.5° C.
 149. Thermocyclerapparatus according to claim 148 wherein said pressing cover comprises aheated platen maintainable at a temperature in the range of 94°-110° C.150. Thermocycler apparatus according to claim 148 wherein saidmultizone heating system comprises a film heater in thermal contact withthe bottom surface of the sample block.
 151. Thermocycler apparatusaccording to claim 150 wherein said bias cooling system comprises aseries of bias cooling channels through said block parallel to said topsurface and parallel to and spaced from the rows of wells, and pumpmeans for pumping cooling liquid through said bias cooling channels.152. Thermocycler apparatus according to claim 151 wherein said rampcooling system comprises a series of ramp cooling channels through saidblock parallel to the bias cooling channels and spaced apart therefromand from the rows of wells, and pump means for pumping cooling liquidthrough said ramp cooling channels, entering at opposite ends ofsuccessive ramp cooling channels.
 153. Thermocycler apparatus accordingto claim 152 wherein there is one bias cooling channel and one rampcooling channel proximate each row of sample wells.
 154. Thermocyclerapparatus according to claim 148 further comprisingh. a two-pieceplastic holder for loosely holding up to 96 microtiter sample tubes of apreselected design, each having a cylindrically shaped upper sectionopen at its top end and a closed, tapered lower section extendingdownwardly therefrom, each tube being of circular cross section andhaving a circumferential shoulder extending outwardly from said uppersection at a position on said upper section spaced from the open endthereof, comprisingaa. a one-piece tray member comprisingi. a flat,horizontal plate section containing 96 holes in an 8-by-12 rectangulararray compatible with industry standard microtiter plate format, saidholes being slightly larger than the outside diameter of the uppersections of said tubes but smaller than the outside diameter of saidshoulder, ii. a first vertical tray sidewall section completely aroundsaid plate extending upwardly to a height greater than the height of atube resting in one of said holes, iii. a second vertical tray sidewallsection around said plate extending downwardly approximately to thebottom of the upper section of a tube resting in one of said holes, bb.a one-piece retainer releasably engageable inside said tray over anysample tubes resting in said tray comprisingi. a flat, horizontal platesection containing 96 holes in an 8-by-12 rectangular array compatiblewith industry standard microtiter plate format, said holes beingslightly larger than the outside diameter of the upper sections of saidtubes but smaller than the outside diameter of said shoulder, ii. avertical retainer sidewall section around said retainer plate sectionextending upwardly from said plate,wherein when said retainer is engagedinside said tray, the retainer plate section lies slightly above theshoulder of a tube resting in said tray and the first tray sidewallsection is about as high as said retainer sidewall section, wherebytubes resting in said tray are retained loosely both vertically andlaterally and extend downwardly into said sample wells, i. up to 96microtiter sample tubes in said holder, each of which engages adeformable cap for forming a gas-tight seal thereon, and each of whichsaid caps protrudes slightly above an uppermost edge of said two-pieceplastic holder when said sample tubes are seated in said holder and insaid sample wells.
 155. Thermocycler apparatus according to claim 154wherein the downward displacement of said cover deforms the tops of saidcaps downwardly until the displacement is stopped by said uppermost edgeof said two-piece plastic holder.
 156. Thermocycler apparatus accordingto claim 155 wherein said uppermost edge of said two-piece plasticholder contacts the underside of said cover around the entire peripheryof said edge, thus forming a gas-tight seal.
 157. Thermocycler apparatusaccording to claim 148 comprising at least two heating zones for theguard band.
 158. The apparatus of either of claims 1 or 150, furthercomprising said tube wherein said tube comprises a PCRreaction-compatible one-piece plastic microcentrifuge-type tubecomprising a first substantially cylindrically shaped upper wall sectionand a second substantially conically shaped lower wall sectioncharacterized by said second wall section being substantially reduced inthickness relative to said first wall section to a thickness of no morethan approximately 0.012 inches, wherein substantially entirely saidsecond wall section contacts the wall of said well.
 159. The apparatusof either of claims 1 or 142, further comprising said tube wherein saidtube comprises a PCR reaction-compatible one-piece plasticmicrocentrifuge-type tube comprising a first substantially cylindricallyshaped upper wall section and a second substantially conically shapedlower wall section characterized by said second wall section beingsubstantially reduced in thickness relative to said first wall sectionto a thickness of no more than approximately 0.012 inches, whereinsubstantially entirely said second wall section contacts the wall ofsaid well and further comprising a cap wherein said cap is of one pieceand is pivotally attached to said tube and removably insertable intosaid tube to hermetically seal it, said cap comprising a substantiallycylindrically shaped third lower section, a resiliently deformableclosed fourth top section and a circumferential shoulder, extendingradially outwardly, attached to said third section.
 160. In acombination of a thermal cycler for performing a polymerase chainreaction comprising a temperature-controlled metal block having an arrayof tapered wells in its top surface, and a plurality of individualreaction tubes having similarly tapered lower sections and uppersections which project above the top surface of the block when the tubesare placed in said wells, the improvement comprising means for seatingsaid tubes in said wells, said seating means comprising:a) resilientlydeformable sealing caps removably attached to said tubes, b) a platen,c) support means for supporting the platen above the block, and d)displacement means associated with the support means for raising theplaten above said caps and for lowering it so as to apply a force of atleast 30 grams to each cap.
 161. The apparatus according to claim 160further comprising controllable heating means to maintain said platen ata preselected temperature in the range of 94°-100° C.
 162. The apparatusaccording to claim 161 further comprising skirt means attached to saidsupport means for isolating said block and said tubes.
 163. Theapparatus according to claim 161, wherein said displacement meanscomprises screw means.
 164. The apparatus according to claim 161 furthercomprising a two-piece plastic holder for loosely holding up to 96 ofsaid tubes, each tube having a cylindrically shaped upper section openat its top end and a closed, tapered lower section extending downwardlytherefrom, each tube also being of circular cross section and having acircumferential shoulder extending outwardly from said upper section ata position on said upper section spaced from the open end thereof,comprisinga one-piece tray member comprisingi. a flat, horizontal platesection containing 96 holes in an 8-by-12 rectangular array compatiblewith industry standard microtiter plate format, said holes being.Slightly larger than the outside diameter of the upper sections of saidtubes but smaller than the outside diameter of said shoulder. ii. afirst vertical tray sideway section completely around said plateextending upwardly to a height greater than the height of a tube restingin one of said holes, and iii. a second vertical tray sidewall sectionaround said plate extending downwardly approximately to the bottom ofthe upper section of a tube resting in one of said holes, and b. aone-piece retainer releasably engageable inside said tray over anysample tubes resting in said tray comprisingi. a flat, horizontal platesection containing 96 holes in an 8-by-12 rectangular array compatiblewith industry standard microtiter plate format, said holes beingslightly larger than the outside diameter of the upper sections of saidtubes but smaller than the outside diameter of said shoulder, and ii. avertical retainer sidewall section around said retainer plate sectionextending upwardly from said plate,wherein when said retainer is engagedinside said tray, the retainer plate section lies slightly above theshoulder of a tube resting in said tray and the first tray sidewallsection is about as high as said retainer sidewall section, wherebytubes resting in said tray are retained loosely both vertically andlaterally and wherein said holes in said two-piece plastic holdergenerally align with said wells in said metal block when said two-pieceplastic holder is in place on said metal block.
 165. The apparatusaccording to claim 164, wherein said displacement means includes meansfor lowering said platen so as to apply a force of 50-100 grams to eachcap.
 166. The apparatus of claim 164, wherein said displacement meansfurther comprises means for seating said platen on said uppermost edgeof said holder such that said tubes are substantially enclosed by theblock, the uppermost edge of said holder and the platen.
 167. Theapparatus of claim 160, wherein said plurality of reaction tubes areloosely held in an array.